Mouse artificial chromosome vector

ABSTRACT

Disclosed is a mouse artificial chromosome vector, comprising: a natural centromere derived from a mouse chromosome; a mouse-chromosome-derived long-arm fragment formed by deleting a long-arm distal region at a mouse chromosome long-arm site proximal to the centromere; and a telomere sequence, wherein the vector is stably retained in a cell and/or tissue of a mammal. In addition, disclosed are cells or non-human animals comprising the vector, and use of the cells or non-human animals.

TECHNICAL FIELD

The present invention relates to mouse artificial chromosome vectorsthat can be stably retained in vivo in a rodent and can be transmittedto offspring.

The present invention also relates to cells comprising the above mouseartificial chromosome vector.

The present invention further relates to non-human animals, such as amouse, comprising the above mouse artificial chromosome vector.

BACKGROUND ART

A transgenic mouse, which is produced by introducing a gene-carryingvector, has been widely used to utilize a gene of interest and itsexpression product. Unfortunately, in conventional transgenic mice, atransgene is introduced into any site at random, and the positionaleffect of the insertion site may cause reduced expression of thetransgene. In addition, conventional gene transfer methods can notcontrol the copy number of a transgene, and limit the size of atransgene to about 200 kb. Due to these problems, it was difficult toclone a gene or gene cluster of more than 200 kb which is not uncommonto mammalian genes, optionally comprising a regulatory region, into avector. In the conventional gene transfer methods, intrinsic functionsof a transgene could not thus be reconstituted and examined, whichsituation has set a limit.

In the presence of such a problem, the present inventors have developeda technique for producing a transchromosomic mouse by using a novelchromosomal transfer method that introduces genes at a chromosomal level(Non-Patent Literature 1). This technique has allowed a human chromosomeor a fragment thereof to be introduced into a mouse embryonic stem (ES)cell, whereby chimeric mice have been produced. This study demonstratedthat the human chromosome fragment has been independently retained in EScells; a plurality of human genes have been expressed in atissue-specific manner; and some human chromosomes have been able to bepartially transmitted to offspring after having undergone meiosis. Thepresent inventors also have introduced the entire human chromosome 21(about 35 Mb) into a mouse, and have created a Down syndrome model mousehaving a high practical value (Non-Patent Literature 2). Analysis ofthis mouse has revealed effectiveness of the chromosome vector becausethe mouse has exhibited the physiological expression pattern of thegenes of the introduced human chromosome 21.

The techniques the present inventors have employed further includechromosome engineering procedures such as a chromosome deletion methodusing a telomere truncation technique utilizing an artificial telomeresequence and a chromosome cloning method using a Cre/loxP system. Thesemethods have allowed for construction of a human artificial chromosomecontaining only a target region. As a result, the present inventors havesuccessfully constructed a human artificial chromosome (HAC) vectorcontaining a specific human chromosome region having a size of megabases (Mb), and have demonstrated that the vector functions in a mouseindividual (Non-Patent Literature 3). Furthermore, the above techniqueshave been used to construct a novel HAC vector without known genes(Non-Patent Literature 4). In addition, based on the above background,the present inventors have successfully achieved stable expression of agene of interest by introducing into any cell a HAC vector carrying thegene of interest. Additional examples of a humanized model mousecarrying the HAC vector have been produced as follows: adrug-metabolizing enzyme CYP3A gene cluster (1 Mb) of human chromosome 7and a human DMD gene (2.5 Mb) responsible for human X-linked musculardystrophy have been each cloned into a HAC vector (CYP3A-HAC, DMD-HAC);and these vectors have been each introduced into a mouse ES cell toproduce mice (Patent Literature 1, Non-Patent Literature 5).

A tissue retention rate and expression analysis of the mouse having theCYP3A-HAC have demonstrated that the CYP3A gene cluster on the HAC hasbeen retained in each tissue of the mouse (FIG. 8, Patent Literature 1).Its expression pattern has been similar to that of a human tissuecounterpart. That is, the expression pattern has been specific to aliver and a small intestine. In addition, a tissue retention rate andexpression analysis of the mouse having the DMD-HAC have demonstratedthat the DMD-HAC has been retained in each tissue of the mouse (FIG. 4A,Non-Patent Literature 5). The mouse has expressed, like a human, atleast three splicing isoforms known to be expressed in a tissue-specificmanner in a human. This series of results suggest usefulness of theHAC-carrying mouse as a novel gene (gene group)-transfer alternative fora conventional transgenic mouse.

Mammalian artificial chromosome vectors, including a human artificialchromosome, have advantages that conventional vector systems (e.g., avirus, a YAC, a BAC, a PAC, a cosmid, and a plasmid) do not have. Thus,the mammalian artificial chromosome vectors should be useful as a systemfor analyzing functions of a novel gene and for generating a humanizedmodel animal. For example, Patent Literatures 2 and 3 disclose HACvectors in which human chromosome 14 or 21 was modified; the chromosomewas reduced in size to yield a fragment; and the fragment was relativelystably retained in cells.

Unfortunately, with regard to the human chromosome 21 transferred mouse(i.e., a Down syndrome model mouse) or the HAC vector transferred mouse,which enables the introduction of a gene of Mb units that was impossiblefor conventional genetically modified mice, there exist at least thefollowing problems: that is, the human chromosome vector has a decreasedretention rate; the retention rate varies among tissues and individuals;and the frequency of transmission to offspring is not stable. Thisfacilitates the need to always consider the retention rate of the HACvectors. Further, when the involvement with functions of a specific generegion or diseases is studied, there is a case where it may be difficultto analyze in detail and precisely a mode of expression of a gene ofinterest and its expression product at a tissue and/or cellular level.These things will constitute a barrier to highly reproducible, uniformanalysis.

Moreover, in the case of conducting the cell fusion between a mouse celland a human cell, a human chromosome is known to be unstable in themouse cell. Because the human chromosome, including a human artificialchromosome vector, has thus a variable retention rate in the mouse cell,when the human artificial chromosome vector is introduced into a mousecell to generate a transgenic mouse, the human artificial chromosomevector does not exhibit full advantages as an artificial chromosomevector. When a mouse cell having a transgene or a transgenic mouse isgenerated, the retention rate of the transgene should be improved and bemade constant. This can promise more detailed, precise, highlyreproducible gene function analysis or effective recovery of theexpression product of the transgene.

PRIOR ART LITERATURES Patent Literatures

-   Patent Literature 1: WO 2009/063722-   Patent Literature 2: WO 2004/031385-   Patent Literature 3: Japanese Patent Publication (Kokai) No.    2007-295860A

Non-Patent Literatures

-   Non-Patent Literature 1: Tomizuka et al., Nat Genet, 16, 133-143,    1997-   Non-Patent Literature 2: Shinohara et al., HMG, 10, 1163-75, 2001-   Non-Patent Literature 3: Kuroiwa et al., Nat Biotech, 18, 1086-1090,    2000-   Non-Patent Literature 4: Katoh et al., BBRC, 321, 280-290, 2000-   Non-Patent Literature 5: Hoshiya et al., Mol Ther, 17, 309-17, 2009

SUMMARY OF INVENTION Problem to be Solved by Invention

Whereas many of previously reported mammalian artificial chromosomes arehuman artificial chromosomes (JP Patent Publication (Kokai) No.2005-230020A; No. 2008-54501A; No. 2007-306928A; and No. 2007-295860A),there are a few reports on mouse artificial chromosomes, wherein theseartificial chromosomes are characterized by using a sequence derivedfrom a portion of a mouse centromere (S. Stewart et al. (2002) GeneTherapy 9, 719-723).

When a natural human chromosome fragment is transferred into a mousecell, the human chromosome fragment is unstable in the mouse cell aspreviously described (e.g., Shinohara et al. (2000) Chromosome Research,8, 713-725). This unstability is also similar even in the body and eachtissue of a mouse individual. The proportion (or retention rate) of thehuman artificial chromosome retained in the mouse tissues and cellstends to decrease, and the retention rate in the mouse tissues and cellsbecomes variable. The same applies to murine individuals. That is, theretention rate of the human artificial chromosome becomes variable amongmurine individuals. Such a non-constant retention rate among the murinetissues and individuals causes difficulties in performing detailed,highly reproducible analysis on a transgene by using a murine cell orindividual having a target gene (or a group of genes) introduced byusing a human artificial chromosome.

As described above, few reports have disclosed artificial chromosomesderived from rodents including mouse and related matters. In addition,there are no artificial chromosomes that are kept stable in a rodentcell or individual. Thus, an object of the present invention is toprovide a mouse artificial chromosome vector in which a transgene (agroup of transgenes) of interest is/are kept stable in a rodent cell orindividual, thereby enabling detailed, precise, highly reproducibleanalysis.

Means for Solving Problem

As a summary, the present invention includes the following features.

(1) A mouse artificial chromosome vector, comprising: an naturallyoccurring centromere derived from a mouse chromosome; amouse-chromosome-derived long-arm fragment formed by deleting a long-armdistal region at a mouse chromosome long-arm site proximal to thecentromere; and a telomere sequence, wherein the vector is stablyretained in a cell and/or tissue of a mammal.(2) The mouse artificial chromosome vector according to the above aspect(1), wherein the mouse chromosome is any one of chromosomes 1 to 19.(3) The mouse artificial chromosome vector according to the above aspect(1) or (2), wherein the mouse-chromosome-derived long-arm fragment isthe remainder region formed by deleting a region including at least99.5% of all endogenous genes from a long arm of any one of mousechromosomes 1 to 19.(4) The mouse artificial chromosome vector according to any of the aboveaspects (1) to (3), wherein the vector comprises, as a basic structure,a mouse artificial chromosome contained in the deposited cell line DT40B6bT-1 (FERM BP-11128).(5) The mouse artificial chromosome vector according to any of the aboveaspects (1) to (4), wherein the mammal is a rodent.(6) The mouse artificial chromosome vector according to the above aspect(5), wherein the rodent is a mouse, rat, or hamster.(7) The mouse artificial chromosome vector according to any of the aboveaspects (1) to (6), further comprising one or more DNA sequenceinsertion sites.(8) The mouse artificial chromosome vector according to the above aspect(7), wherein the DNA sequence insertion site is a recognition site for asite-specific recombinase.(9) The mouse artificial chromosome vector according to the above aspect(7) or (8), wherein the DNA sequence insertion site is a loxP sequence,an FRT sequence, φC31 attB and φC31 attP sequences, R4 attB and R4 attPsequences, TP901-1 attB and TP901-1 attP sequences, or Bxb1 attB andBxb1 attP sequences.(10) The mouse artificial chromosome vector according to any of theabove aspects (1) to (9), further comprising a reporter gene, aselection marker gene, or both.(11) The mouse artificial chromosome vector according to any of theabove aspects (1) to (10), further comprising an exogenous DNA sequence.(12) The mouse artificial chromosome vector according to any of theabove aspects (1) to (11), wherein the exogenous DNA sequence has a sizeof 200 kb or more.(13) The mouse artificial chromosome vector according to the aboveaspect (11) or (12), wherein the exogenous DNA sequence is a human DNAsequence.(14) The mouse artificial chromosome vector according to any of theabove aspects (11) to (13), wherein the exogenous DNA sequence is a DNAsequence of a drug-metabolism-related gene.(15) The mouse artificial chromosome vector according to the aboveaspect (14), wherein the drug-metabolism-related gene is a gene encodingan enzyme involved in a phase I reaction or a phase II reaction.(16) The mouse artificial chromosome vector according to the aboveaspect (15), wherein the enzyme gene involved in a phase I reactionencodes at least one enzyme selected from the group consisting of CYPssuch as CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A,CYP4A, CYP4B, and subfamilies thereof, and CESs.(17) The mouse artificial chromosome vector according to the aboveaspect (15), wherein the enzyme gene involving the phase II reactionencodes at least one enzyme selected from the group consisting of UGT1and UGT2.(18) The mouse artificial chromosome vector according to the aboveaspect (14), wherein the drug-metabolism-related gene is a gene encodinga transporter.(19) The mouse artificial chromosome vector according to the aboveaspect (18), wherein the gene encoding the transporter is at least onegene selected from the group consisting of MDR1, MDR2, MRP2, OAT, OATP,OCT, and BCRP.(20) The mouse artificial chromosome vector according to the aboveaspect (14), wherein the drug-metabolism-related gene is a gene encodinga nuclear receptor.(21) The mouse artificial chromosome vector according to the aboveaspect (20), wherein the gene encoding the nuclear receptor is at leastone gene selected from the group consisting of PXR, AhR, CAR, and PPARα.(22) The mouse artificial chromosome vector according to any of theabove aspects (11) to (13), wherein the exogenous DNA sequence is a DNAsequence of a human-chromosome-derived long arm or short arm.(23) The mouse artificial chromosome vector according to any of theabove aspects (11) to (21), wherein the exogenous DNA sequence comprisesat least two genes selected from the group consisting of genes encodingan enzyme involved in a phase I reaction, genes encoding an enzymeinvolved in a phase II reaction, genes encoding a transporter and genesencoding a nuclear receptor.(24) The mouse artificial chromosome vector according to the aboveaspect (22), wherein the DNA sequence of the human-chromosome-derivedlong arm or short arm comprises a human chromosome region responsiblefor a disease gene.(25) The mouse artificial chromosome vector according to any of theabove aspects (11) to (13), wherein the exogenous DNA sequence is a geneor DNA sequence encoding a polypeptide such as cytokines, hormones,growth factors, nutritional factors, hematopoietic factors, coagulationor hemolysis factors, immunoglobulins, G protein-coupled receptors, orenzymes, or a gene or DNA sequence used for treatment involved in adisease such as tumor, muscular dystrophy, hemophilia, neurodegenerativedisease, autoimmune disease, allergic disease, or genetic disease.(26) The mouse artificial chromosome vector according to any of theabove aspects (1) to (25), wherein the cell is a hepatocyte, enterocyte,renal cell, splenocyte, lung cell, cardiac cell, skeletal muscle cell,brain cell, bone marrow cell, lymphocyte, megakaryocyte, sperm, or ovum.(27) The mouse artificial chromosome vector according to any of theabove aspects (1) to (25), wherein the tissue is derived from a liver,intestine, kidney, spleen, lung, heart, skeletal muscle, brain, bonemarrow, testis, or ovary.(28) A cell comprising the mouse artificial chromosome vector accordingto any of the above aspects (1) to (27).(29) The cell according to the above aspect (28), wherein the cell isselected from the group consisting of somatic cells, non-human germ-linecells, stem cells, and precursor cells.(30) The cell according to the above aspect (29), wherein the stem cellis an embryonic stem (ES) cell or an induced pluripotent stem (iPS)cell.(31) The cell according to any of the above aspects (28) to (30),wherein the cell is a primary cultured cell, subcultured cell, or cellline.(32) The cell according to any of the above aspects (28) to (31),wherein the cell is capable of producing a human antibody.(33) A pharmaceutical composition comprising the cell according to anyof the above aspects (28) to (32), wherein the cell comprises a mouseartificial chromosome vector comprising an exogenous DNA sequence foruse in treating a disease.(34) A non-human animal comprising the mouse artificial chromosomevector according to any of the above aspects (1) to (27).(35) The non-human animal according to the above aspect (34), whereinthe animal is a disease-model animal.(36) The non-human animal according to the above aspect (34), whereinthe animal is capable of expressing an exogenous humandrug-metabolism-related gene.(37) The non-human animal according to the above aspect (34), whereinthe animal is capable of producing a human antibody.(38) The non-human animal according to any of the above aspects (34) to(37), wherein an endogenous gene corresponding to an exogenous DNAcontained in the mouse artificial chromosome vector is disrupted or theendogenous gene has decreased expression.(39) A process for producing a protein, comprising: culturing the cellaccording to any of the above aspects (28) to (32), the cell comprisingthe mouse artificial chromosome vector comprising a sequence of anexogenous DNA to produce a protein encoded by the DNA; and collectingthe protein.(40) A process for producing a human antibody, comprising: using thenon-human animal according to the above aspect (37) or (38), the animalcomprising the mouse artificial chromosome vector comprising a geneencoding a human antibody to produce the human antibody; and collectingthe human antibody.(41) A method for screening for a substance effective in treating adisease, comprising: administering a candidate drug to a disease-modelanimal of the non-human animal according to the above aspect (35) as adisease-model animal; and evaluating a therapeutic effect of the drug.(42) A method for testing a pharmacological effect and/or metabolismand/or toxicity of a drug or food, comprising: administering a drug orfood to the non-human animal according to the above aspect (36) or (38)or a cell, organ, or tissue thereof, wherein the animal, cell, organ ortissue comprises the mouse artificial chromosome vector comprising ahuman drug-metabolism-related gene; and determining a pharmacologicaleffect and/or metabolism and/or toxicity of the drug or food.(43) A method for testing toxicity of a drug or food, comprising:coculturing a drug and/or food and a culture cells or bacterium with amicrosome or S9 microsome fraction as obtained from the non-human animalaccording to the above aspect (36) or (38), wherein the animal comprisesthe mouse artificial chromosome vector comprising a humandrug-metabolism-related gene; and determining an effect of the drug orfood on the culture cell or bacterium.(44) A method for stabilizing a large-size DNA in a cell or individual,comprising: using the mouse artificial chromosome vector according toany of the above aspects (1) to (27) to stably keep an exogenous DNAhaving a large size of 200 kb or more in a rodent cell or individual ata retention rate of 90% or more.

According to the present invention, when a gene (or a group of genes) ofinterest is/are introduced into a rodent cell or individual, the mouseartificial chromosome vector comprising a DNA sequence insertion sitecan maintain the gene (or a group of genes) stably and at an identicalretention rate in any cell or tissue, although it was difficult tointroduce the gene or a group of genes into the cell or tissue byconventional means, and because a reporter gene can be inserted togetherwith an exogenous DNA sequence or gene of interest, the cell carryingthe vector can be visualized and detailed, precise, highly reproducibleanalyses or effective recovery of expression products can be achieved.

The contents described in the specification and/or drawings of JapanesePatent Application No. 2010-1425, from which the present applicationclaims the priority, are herein incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

The application contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee.

FIG. 1 is a schematic diagram illustrating the procedures of Examples 1and 2 later. Cell names in the figure are described according to thefollowing format. Cell name (cellular genetic modification; retainedchromosome fragment name, and transgene-retaining chromosome name).Symbols given in the drawings are as follows. BSr: blasticidin (BS)resistant gene, puro: puromycin resistant gene, artificial telomere:artificial telomere (TTAGGG) repeat sequence, EGFP: gene expressingenhanced green fluorescent protein, neo: neomycin (G418) resistant gene,loxP: site specific DNA sequence insertion site, 3′ HPRT: the 3rd to 9thexon sequences of HPRT gene.

FIG. 2 is a schematic diagram illustrating the procedures of Example 3.Cell names in the drawings are described according to the followingformat. Cell name (cellular genetic modification; retained chromosomefragment name, and transgene-retaining chromosome name). Symbols givenin the figure are as follows. hChr7: human chromosome 7, CYP3A cluster:human CYP3A gene cluster, 5′ HPRT: theist to 2nd exon sequences of HPRTgene, loxP: site specific DNA sequence insertion site, hyg: hygromycinresistant gene, hisD: histidinol resistant gene, artificial telomere:artificial telomere (TTAGGG) repeat sequence, EGFP: gene expressingenhanced green fluorescent protein, neo: neomycin (G418) resistant gene,3′ HPRT: the 3rd to 9th exon sequences of HPRT gene, puro: puromycinresistant gene.

FIG. 3 is a schematic diagram showing the procedures of Example 4. Cellnames in the drawings are described according to the following format.Cell name (cellular genetic modification; retained chromosome fragmentname, and transgene-retaining chromosome name). Symbols given in thedrawings are as follows. puro: puromycin resistant gene, artificialtelomere: artificial telomere (TTAGGG) repeat sequence, 5′ HPRT: the 1stto 2nd exon sequences of HPRT gene, hyg: hygromycin resistant gene,loxP: site specific DNA sequence insertion site.

FIG. 4 is a schematic diagram illustrating the procedures of Example 5.Cell names in the drawings are described according to the followingformat. Cell name (cellular genetic modification; retained chromosomefragment name, and transgene-retaining chromosome name). Symbols givenin the drawings are as follows. puro: puromycin resistant gene,artificial telomere: artificial telomere (TTAGGG) repeat sequence, neo:neomycin (G418) resistant gene, loxP: site specific DNA sequenceinsertion site, 3′ HPRT: the 3rd to 9th exon sequences of HPRT gene.

FIG. 5 is a schematic diagram illustrating the procedures of Example 6.Cell names in the drawings are described according to the followingformat. Cell name (cellular genetic modification; retained chromosomefragment name, and transgene-retaining chromosome name). Symbols givenin the drawings are as follows. neo: neomycin (G418) resistant gene,loxP: site specific DNA sequence insertion site, 3′ HPRT: the 3rd to 9thexon sequences of HPRT gene, puro: puromycin resistant gene, artificialtelomere: artificial telomere (TTAGGG) repeat sequence, EGFP: geneexpressing enhanced green fluorescent protein, 5′ HPRT: the 1st to 2ndexon sequences of HPRT gene.

FIG. 6 shows the mouse A9 cell (mouse A9 (neo)) (left) and the cellfusion clone (mouse A9× mouse embryonic fibroblast hybrid (neo;mChr11-BSr)) between the mouse A9 cell and mouse fibroblast (mouseembryonic fibroblast (mChr11-BSr)).

FIG. 7 shows the results of FISH analysis of DT40 (mChr11-Bsr) clone inwhich mouse Cot-1 DNA is used as a probe.

FIG. 8 shows the results of the SKY FISH analysis indicating that mousechromosome (mChr11-BSr) introduced into the chicken DT40 cell is mousechromosome 11 (left panel), and the SKY FISH staining image (rightpanel).

FIG. 9 shows the vector for telomere truncation in AL671968 region ofmouse chromosome 11 and a partial structure of the mouse chromosome 11allele, in which homologous recombination has been carried out by usingthe vector.

FIG. 10 shows the results of mono-color FISH analysis of DT40 (MAC)[DT40 (B6bT-1)] clone containing allele of mouse artificial chromosomeMAC in which telomere truncation has occurred at AL671968 region ofmouse chromosome 11 by using pBS-TEL/puro_MAC vector (right panel). DT40(mChr11-BSr) of the left panel indicates DT40 (mChr11-BSr) clone beforethe telomere truncation.

FIG. 11 shows a GFP-neo-loxP-3′ HPRT type of loxP targeting vector(pMAC1) and a partial structure of allele of mouse artificial chromosomeMAC in which homologous recombination has been carried out by using thevector.

FIG. 12 shows the results of the two-color FISH analysis of DT40 (MAC1)clone in which mouse Cot-1 DNA and GFP-PGKneo-loxP-3′ HPRT cassette wereused as probes.

FIG. 13 shows the results of the mono-color FISH analysis of CHO(HPRT;MAC1) clone in which mouse Cot-1 DNA was used as a probe.

FIG. 14 shows the results of the mono-color FISH analysis of mouse ES(MAC1) clone in which mouse minor satellite DNA was used as a probe.

FIG. 15 shows the targeting vector (pMPloxPHyg) for inserting loxP intothe AC004922 region, which locates extremely close to CYP3A gene locusof human chromosome 7 and on the centromere side (i.e., locating on thecentromere side by approximately 300 Kb from CYP3A gene locus), and apartial structure of the human chromosome 7 allele in which homologousrecombination has been carried out by using the vector (FIG. 15 a). FIG.15 b shows the analysis results of homologous recombinants usingSouthern hybridization with respect to hygromycin resistant cell linesof DT40 cell clone containing human chromosome 7 fragment which has beentransfected with the linearized vector. With regard to the arrowheads ofFIG. 15 b, the top arrowhead indicates the non-homologous recombinant(approximately 10.9 kb) and the bottom arrowhead indicates thehomologous recombinant (approximately 8.9 kb).

FIG. 16 shows the targeting vector (pTELhisD-PT) for inserting the humantelomere sequence into the AC073842 region, which is located extremelyclose to CYP3A gene locus of human chromosome 7 and at the telomere side(i.e., locating on the telomere side by approximately 150 Kb from CYP3Agene locus), and a partial structure of the human chromosome 7 allele inwhich homologous recombination has been carried out by using the vector.

FIG. 17 shows the results of the two-color FISH analysis of CHO(HPRT-,MAC1+hChr7-loxP-tel) clone in which mouse Cot-1 DNA and human Cot-1 DNAwere used as probes.

FIG. 18 shows the construction of mouse artificial chromosome CYP3A-MACby translocation-cloning approximately 1 Mb human CYP3A gene clusterregion (i.e., AC004922-human CYP3A gene cluster-AC073842) into MAC1.

FIG. 19 shows the results of the two-color FISH analysis of CHO(CYP3A-MAC, hChr7-ΔCYP3A) clone in which human Cot-1 DNA and mouse Cot-1DNA were used as probes.

FIG. 20 shows the targeting vector (pMAC2) for constructing the mouseartificial chromosome vector MAC2, and a partial structure of allele ofmouse artificial chromosome MAC in which homologous recombination hasbeen carried out by using the vector.

FIG. 21 shows the results of the two-color FISH analysis of DT40 (MAC2)clone in which mouse Cot-1 DNA and 5′ HPRT-loxP-PGK hygro cassette wereused as probes.

FIG. 22 shows the results of the two-color FISH analysis of CHO(HPRT⁻;MAC2) clone in which mouse Cot-1 DNA and 5′ HPRT-loxP-PGK hygro cassettewere used as probes.

FIG. 23 shows the results of the mono-color FISH analysis of mouse ES(HPRT-, MAC2) clone in which mouse minor satellite DNA was used as aprobe.

FIG. 24 shows a PGKneo-loxP-3′ HPRT type of loxP targeting vector(pMAC3), and a partial structure of allele of mouse artificialchromosome MAC3 in which homologous recombination has been carried outby using the vector.

FIG. 25 shows the results of the two-color FISH analysis of DT40 (MAC3)clone in which mouse Cot-1 DNA and mouse minor satellite DNA were usedas probes.

FIG. 26 shows the results of the mono-color FISH analysis of CHO(HPRT⁻;MAC3) clone in which mouse Cot-1 DNA was used as a probe.

FIG. 27 shows the results of the mono-color FISH analysis of drugresistant clone B6 (HPRT⁻; MAC3) in which mouse minor satellite DNA wasused as a probe.

FIG. 28 shows the results of the analysis of retention rate duringlong-term culture of B6 (HPRT⁻; MAC3) clone. The solid line and thedashed line indicate the MAC vector retention rates in B6 (HPRT⁻:MAC3)-3 and B6 (HPRT⁻: MAC3)-s6, respectively.

FIG. 29 shows the procedure for performing the site-specific geneinsertion of a certain gene (for example, GFP) into the mouse artificialchromosome vector MAC3 by Cre-loxP method, a vector for inserting GFP,and a partial structure of the mouse chromosome 11 allele in whichhomologous recombination was carried out by using the vector.

FIG. 30 shows the results of the two-color FISH analysis of CHO(GFP-MAC) clone, which is a CHO cell retaining the mouse artificialchromosome GFP-MAC, in which mouse Cot-1 DNA and X6.1 EGFP were used asprobes.

FIG. 31 shows the results of the FISH analysis of the mouse artificialchromosome GFP-MAC after long-term culture of mouse ES cells (B6-ES cellline), in which mouse minor satellite DNA and GFP were used as probes.

FIG. 32 shows the results of the analysis of retention rate duringlong-term culture of B6 (GFP-MAC) clone. The diamond represents theretention rate for the long-term culture in which drug selection wasperformed, while the square represents the retention rate for the longterm culture in which no drug selection was performed.

FIG. 33 shows a progeny transfer individual that was born from achimeric mouse retaining the mouse artificial chromosome vector(GFP-MAC).

FIG. 34 shows the retention rate of 21HAC1 or 21HAC2 and MAC1 in CHOcells after long-term culture (25 PDL).

FIG. 35 shows the retention rate of 21HAC2 or MAC1 in ES cells afterlong-term culture (75 PDL).

FIG. 36 shows the stereo fluorescent microscopic images of TC (MAC1)mouse (female) tissues.

FIG. 37 shows the GFP positive rate in hematopoietic cells of bonemarrow-derived cells of TC (MAC1) mouse or TC (21HAC2) mouse.

FIG. 38 shows the GFP positive rate in hematopoietic cells ofspleen-derived cells of TC (MAC1) mouse or TC (21HAC2) mouse.

FIG. 39 shows the results of the mono-color FISH analysis of tailfibroblasts derived from TC (MAC1) mouse in which mouse minor satelliteDNA probe was used.

FIG. 40 shows the results of the two-color FISH analysis of A9(CYP3A-MAC) in which CYP3A-BAC(RP11-757A13) and mouse minor satelliteDNA probes were used.

FIG. 41 shows the results of the mono-color FISH analysis of TT2F(CYP3A-MAC) in which CYP3A-BAC(RP11-757A13) DNA probe was used.

FIG. 42 shows the retention rate of CYP3A-MAC in ES cells afterlong-term culture (100 PDL).

FIG. 43 shows stereo fluorescence microscopic images of TC(CYP3A-MAC)mouse (male) tissues.

FIG. 44 shows the GFP positive rate in hematopoietic cells of bonemarrow-derived cells of TC(CYP3A-MAC) mouse or TC(CYP3A-HACΔ) mouse.

FIG. 45 shows the retention rate of CYP3A-MAC or CYP3A-HACA in eachtissue of TC(CYP3A-MAC) mouse or TC(CYP3A-HACΔ) mouse.

FIG. 46 shows the results of the mono-color FISH analysis ofTC(CYP3A-MAC) heterozygous mouse or TC(CYP3A-MAC) homozygous mouse inwhich CYP3A-BAC (RP11-757A13) DNA probe was used.

FIG. 47 shows the results of the analysis of tissue specific geneexpression of CYP3A gene cluster in each tissue of TC(CYP3A-MAC) mouse.GAPDH represents glyceraldehyde 3-phosphate dehydrogenase.

FIG. 48 shows the results of the analysis of time specific geneexpression of CYP3A gene cluster in TC(CYP3A-MAC) mouse liver.

FIG. 49 shows the results of the two-color FISH analysis of rat ES(CYP3A-MAC) in which CYP3A-BAC(RP11-757A13) and mouse Cot-1 DNA probeswere used.

FIG. 50 shows the results of the two-color FISH analysis of CHO(HPRT⁻;MAC1, hChr21-loxP) in which mouse Cot-1 DNA and human Cot-1 DNA probeswere used.

FIG. 51 shows the construction of mouse artificial chromosomehChr21q-MAC in which approximately 33 Mb hChr21q region wastranslocation-cloned into MAC1.

FIG. 52 shows the results of the two-color FISH analysis of CHO(hChr21q-MAC, hChr21-hChr21q) clone in which human Cot-1 DNA and mouseCot-1 DNA were used as probes.

FIG. 53 shows the results of the two-color FISH analysis of TT2F(hChr21q-MAC) clone in which human Cot-1 DNA and mouse minor satelliteDNA probes were used.

FIG. 54 shows the retention rate of hChr21q-MAC in ES cells afterlong-term culture (50 PDL).

FIG. 55 shows the fluorescence picture of a chimeric mouse retaininghChr21q-MAC.

FIG. 56 shows the targeting vector (pCKloxPHyg) for inserting loxPsequence into the AP001721 proximal to DSCR (Down's syndrome causativeregion cluster) of human chromosome 21 (hChr21), a target sequence, anda chromosome allele produced by homologous recombination.

FIG. 57 shows the results of the Southern blot analysis of DT40(hChr21q22.12-loxP).

FIG. 58 shows the results of the two-color FISH analysis of DT40(hChr21q22.12-loxP) clone in which human Cot-1 DNA and hygromycin DNAwere used as probes.

FIG. 59 shows the results of the two-color FISH analysis of CHO(HPRT⁻;MAC1, hChr21q22.12-loxP) clone in which human Cot-1 DNA and mouse Cot-1DNA were used as probes.

FIG. 60 shows the construction of mouse artificial chromosomehChr21q22.12-MAC in which approximately 12 Mb hChr21q22.12-qter regionis translocation-cloned into MAC1.

FIG. 61 shows the results of the two-color FISH analysis of CHO(hChr21q22.12-MAC, hChr21-hChr21q22.12) clone in which human Cot-1 DNAand mouse Cot-1 DNA were used as probes.

FIG. 62 shows the results of the two-color FISH analysis of TT2F(hChr21q22.12-MAC) clone in which human Cot-1 DNA and mouse minorsatellite DNA probes were used.

FIG. 63 shows the retention rate of hChr21q22.12-MAC in ES cells afterlong-term culture (50 PDL).

FIG. 64 shows the retention rate of 21HAC1 or 21HAC2 and MAC2 in CHOcells after long-term culture (25 PDL).

FIG. 65 shows the structure of one (1) copy-of FVIII-PAC.

FIG. 66 shows the method of constructing 1-16 copies of FVIII-PAC.

FIG. 67 shows the results of the clotting assay (comparison of FVIIIactivity) after long-term culture of CHO (FVIIIx1-MAC)1-3 and CHO(FVIIIx1-HAC)1-2.

FIG. 68 shows the results of the clotting assay (comparison of FVIIIactivity) of CHO (FVIIIx1-MAC) and CHO (FVIIIx16-MAC).

FIG. 69 shows the methods of constructing an entry vector forconstructing a multi-integrase platform cassette.

FIG. 70 shows the method of constructing a multi-integrase platformcassette.

FIG. 71 shows the method of constructing MI-MAC vector.

FIG. 72 shows the method of inserting a gene into MI-MAC vector.

FIG. 73 shows the method of constructing PXR-MAC.

FIG. 74 shows the results of the two-color FISH analysis of CHO(PXR-MAC) clone in which mouse cot-1 DNA and human PXR-BAC-derived DNA(RP11-169N13) (CHORI) were used as probes.

FIG. 75 shows the results of the mono-color FISH analysis of TT2F(PXR-MAC) clone in which human PXR-BAC-derived DNA (RP11-169N13) (CHORI)was used as a probe.

FIG. 76 shows a GFP-5′ HPRT-loxP-hyg type of loxP targeting vector(pMAC4) and a partial structure of mouse artificial chromosome MAC4allele in which homologous recombination was carried out by using thevector.

FIG. 77 shows the results of the two-color FISH analysis of DT40 (MAC4)clone in which mouse cot-1 DNA and GFP-5′ HPRT-loxP-hyg cassette wereused as probes.

FIG. 78 shows the results of the mono-color FISH analysis of CHO(HPRT⁻;MAC4) clone in which mouse Cot-1 DNA was used as a probe.

FIG. 79 shows the targeting vector (pTELpuro-UGT2) for inserting humantelomere sequence into the AC1252392 region, which locates extremelyclose to UGT2 gene locus of human chromosome 4 and on the telomere side(i.e., locating on the telomere side by approximately 150 Kb from UGT2gene locus), and a partial structure of the human chromosome 4 allele inwhich homologous recombination was carried out by using the vector.

FIG. 80 shows the results of the two-color FISH analysis of DT40(hChr4-tel) in which human cot-1 DNA and puromycin DNA were used asprobes. Left panel represents DT40 (hChr4) before modification, andright panel represents DT40 (hChr40-tel) after modification.

FIG. 81 shows a targeting vector (pUGT2loxPneo) for inserting loxPsequence into the AC074378 of human chromosome 4, a target sequence, anda chromosome allele produced by homologous recombination.

FIG. 82 shows the results of the two-color FISH analysis of DT40(hChr4-loxP-tel) in which human cot-1 DNA and neomycin DNA were used asprobes.

FIG. 83 shows the results of the two-color FISH analysis of CHO(HPRT⁻;MAC4, hChr4-loxP-tel) clone in which human Cot-1 DNA and mouse Cot-1 DNAwere used as probes.

FIG. 84 shows the construction of mouse artificial chromosome UGT2-MACin which 2 Mb human UGT2 gene cluster region (i.e., AC074378-human UGT2gene cluster-AC125239) was translocation-cloned into MAC4.

FIG. 85 shows the results of the two-color FISH analysis of CHO(UGT2-MAC, hChr4-ΔUGT2) clone in which UGT2-BAC(RP11-643N16) (CHORI) DNAand mouse Cot-1 DNA were used as probes.

FIG. 86 shows the results of the two-color FISH analysis of A9(UGT2-MAC) clone in which UGT2-BAC(RP11-643N16) (CHORI) and mouse minorsatellite DNA were used as probes.

FIG. 87 shows the results of the mono-color FISH analysis of TT2F(UGT2-MAC) clone in which UGT2-BAC(RP11-643N16) (CHORI) DNA was used asa probe.

FIG. 88 shows the retention rate of UGT2-MAC in ES cells after long-termculture (75 PDL).

FIG. 89 shows a targeting vector (pTELpuro-CYP2C) for inserting a humantelomere sequence into the AL157834 region, which is located extremelyclose to CYP2C gene locus of human chromosome 10 and on the telomereside (i.e., locating on the telomere side by approximately 150 Kb fromCYP2C gene locus), and a partial structure of the human chromosome 10allele in which homologous recombination was carried out by using thevector.

FIG. 90 shows the results of the two-color FISH analysis of DT40(hChr10-tel) in which human cot-1 DNA and puromycin DNA were used asprobes. Left panel represents DT40 (hChr10) before modification, andright panel represents DT40 (hChr10-tel) after modification.

FIG. 91 shows the targeting vector (pCYP2CloxPneo) for inserting loxPsequence into the AL138759 of human chromosome 10, a target sequence,and a chromosome allele produced by homologous recombination.

FIG. 92 shows the results of the two-color FISH analysis of (HPRT⁻;MAC4, hChr10-loxP-tel) clone in which mouse Cot-1 DNA and human Cot-1DNA were used as probes.

FIG. 93 shows the construction of mouse artificial chromosome CYP2C-MACin which 380 kb human CYP2C gene cluster region (i.e., AL138759-humanCYP2C gene cluster-AL157834) was translocation-cloned into MAC4.

FIG. 94 shows the results of the two-color FISH analysis of CHO(CYP2C-MAC, hChr10-ΔCYP2C) clone in which CYP2C-BAC(RP11-466J14) (CHORI)DNA and mouse Cot-1 DNA were used as probes.

FIG. 95 shows the targeting vector (pMDR1loxPbs) for inserting loxPsequence into the AC005045 of human chromosome 7, a target sequence, anda chromosome allele produced by homologous recombination.

FIG. 96 shows a targeting vector (pTELpuro-MDR1) for inserting a humantelomere sequence into the AC003083 region, which is located extremelyclose to MDR1 gene locus of human chromosome 7 and on the telomere side(i.e., locating on the telomere side by approximately 50 Kb from MDR1gene locus), and a partial structure of the human chromosome 7 allele inwhich homologous recombination was carried out by using the vector.

FIG. 97 shows the results of the two-color FISH analysis of DT40(hChr7M-loxP-tel) in which human cot-1 DNA and puromycin DNA were usedas probes.

FIG. 98 shows the results of the two-color FISH analysis of CHO(HPRT⁻;MAC4, hChr7M-loxP-tel) clone in which mouse Cot-1 DNA and human Cot-1DNA were used as probes.

FIG. 99 shows the construction of mouse artificial chromosome MDR1-MACin which 210 kb human MDR1 gene region (i.e., AC005045-human MDR1gene-AC003083) was translocation-cloned into MAC4 vector.

FIG. 100 shows the results of the two-color FISH analysis of CHO(MDR1-MAC, hChr7-ΔMDR1) clone in which MDR1-BAC(RP1′-784L5) (CHORI) DNAand mouse Cot-1 DNA were used as probes.

MODE FOR CARRYING OUT INVENTION

The present invention will be further described in more detail.

As described above, the first embodiment of the present inventionprovides a mouse artificial chromosome vector comprising: an naturalcentromere derived from a mouse chromosome; a mouse-chromosome-derivedlong-arm fragment formed by deleting a long-arm distal region at a mousechromosome long-arm site proximal to the centromere; and a telomeresequence, wherein the vector is stably retained in cells and tissues ofa mammal.

As used herein, the term “natural centromere derived from a mousechromosome” refers to the entire centromere (or the intact centromere)of any one of mouse chromosomes. Thus, the centromere does not include astructure having centromere function which is obtained spontaneously orsynthetically by using a portion of the centromere sequence of a mousechromosome, as well as the centromere of a chromosome derived from otheranimals.

As used herein, the term “mouse artificial chromosome” or “mouseartificial chromosome vector” refers to an artificial chromosomeconstructed by top-down approach, but it does not mean an artificialchromosome constructed by bottom-up approach. The top-down approachrefers to an approach in which gene-coding regions are deleted from anintact chromosome by chromosomal modification; and a natural centromereis used to construct an artificial chromosome vector. The bottom-upapproach refers to an approach in which a portion of a centromeresequence is obtained as a cloned DNA, which is then transfected into amammalian cell to construct an artificial chromosome having centromerefunction.

As used herein, the “mouse-chromosome-derived long-arm fragment formedby deleting a long-arm distal region at a mouse chromosome long-arm siteproximal to the centromere” refers to a long-arm fragment obtained bydeleting a long arm of the mouse chromosome at a long-arm site proximalto the centromere so as to remove the endogenous genes from the long armof the mouse chromosome. This is because it is desirable to eliminateeffects of endogenous genes as possible as, so as to stably keep thevector of the present invention in a mouse cell or tissue and so as notto prevent the development of mice or the genetic transmission tooffspring. This fragment means a long-arm fragment obtained by deletingat least 99.5%, preferably at least 99.7%, more preferably 99.8%, andstill more preferably 99.9% to 100% of total endogenous genes (thenumber of genes) at the long-arm site proximal to the centromere.

As used herein, the term “DNA” can be used to represent any kind of DNAnucleic acid, including a gene or gene locus, cDNA, or chemicallymodified DNA.

As used herein, the term “retention rate,” unless otherwise noted,refers to a rate of cells having an artificial chromosome in a culturecell or a mouse tissue cell.

The term “stably retained” regarding the chromosome vector of thepresent invention means that, during mitosis, the chromosome vector isnot easily dropped-out, that is, even after mitosis, the chromosomevector is stably retained, thereby leading to efficient genetictransmission of the chromosome vector to a daughter cell or a descendantmouse.

The mouse chromosome may be any of mouse chromosomes 1 to 19, X, and Y.The chromosome, however, is preferably any one of chromosomes 1 to 19.Although chromosome 11 is exemplified in Examples below, the otherchromosomes can be used to construct the mouse artificial chromosomevector as long as the above-mentioned characteristics are maintained.

In the case of an artificial chromosome vector derived from a fragmentof mouse chromosome 11, the above long-arm fragment includes, but is notlimited to, a long arm fragment formed by deleting a more distal regionthan, for example, AL671968, BX572640 (locating at a position closer tothe centromere than AL671968), CR954170 (locating at a position closerto the centromere than AL671968 and BX572640), or AL713875 (locating ata position closer to the centromere than AL671968) of the long arm ofthe chromosome 11. Alternatively, the long-arm fragment may comprise, asa basic structure, the mouse artificial chromosome contained in adeposited cell line DT40 B6bT-1 (FERM BP-11128), which chromosome isherein designated as DT40 (MAC) (see FIGS. 1, 3, 4). In addition, in thecase of, for example, an artificial chromosome vector derived from afragment of mouse chromosome 15, the above long-arm fragment includes,but is not limited to, a long-arm fragment formed by deleting a moredistal region than, for example, AC121307 or AC161799. In the case of anartificial chromosome vector derived from a fragment of mouse chromosome16, the above long-arm fragment includes, but is not limited to, along-arm fragment formed by deleting a more distal region at, forexample, AC 127687 or AC 140982. These basic structures (e.g., MAC1,MAC2, MAC3, MAC4; see FIGS. 1, 3, 4) may further comprise a DNA sequenceinsertion site such as loxP to insert an exogenous DNA or gene.

The vector according to the present invention may comprise a sequenceinsertion site for an exogenous DNA or gene. Hence, the integration ofthe exogenous DNA or gene of interest at this site enables the exogenousDNA or gene of interest to be expressed at the time of introduction ofthe vector into a given cell. Thus, the vector (e.g., CYP3A-MAC,GFP-MAC; see FIGS. 2 and 5) is applicable to, for example, proteinproduction, screening for a therapeutic agent, a drug metabolism test,DNA function analysis, iPS cell induction, gene therapy, or generationof a useful non-human animal.

For the vector of the present invention, a mouse chromosome is modifiedand an intact mouse-derived natural centromere is used, therebyconstructing the vector. As a previously known mouse artificialchromosome vector, a satellite DNA-based mammalian artificial chromosome(referred to as Aces or SATAC) which is generated by using a portion ofa centromere sequence is known; however, a mouse artificial chromosomewhich is generated by using the entire centromere of a mouse chromosomedoes not have any precedent. In addition, the above mammalian artificialchromosome, like a HAC vector, has a varied retention rate among tissuesof a mouse individual and is thus unstable (Co Do et al., ChromosomeRes., 2000, 8(3), 83-91).

A useful and unexpected property of the vector of the present inventionis that the retention rate of the vector increases in cells or tissuesof mammals including rodents such as a mouse, rat, and hamster,suggesting that the vector is stably retained in cells, i.e., a gene (agroup of genes) of interest is (are) maintained in cells for a longerperiod. Hence, the amount of a transgene does not vary among rodentindividuals or tissues, and the transgene can be expressed for anextended period. Additional examples of the properties can includeincreased efficiency of genetic transmission and organism development ofa rodent by means of pluripotent cells (e.g., ES cells or iPS cells).Compared with a human artificial chromosome (HAC), the mouse artificialchromosome according to the present invention has intriguing propertiesthat: a variation of the retention rate is extremely small among tissuesincluding hematopoietic tissues in which the retention rate of a HAC isvery low and is less than 20%; i.e., the retention rate is 90% or morein any tissue tested (e.g., tissues derived from the liver, intestine,kidney, spleen, lung, heart, skeletal muscle, brain, or bone marrow).The mouse artificial chromosome of the present invention can alsoproliferate more efficiently than the HAC and can maintain a pluralityof (or multiple) copies in a cell, which is impossible for the HAC.

Definition:

The definitions of terms as used herein specifically include thefollowing meanings in addition to ordinal meanings used in the art.

As used herein, the term “mouse artificial chromosome” or “mouseartificial chromosome vector” refers to an artificial chromosome havingthe above-described characteristics, the artificial chromosome beingconstructed from the mouse-derived chromosome fragment as describedabove. As such, the artificial chromosome is an artificial chromosomeconstructed by top-down approach, not bottom-up approach. Again, thetop-down approach is an approach in which gene regions are deleted froma natural chromosome by chromosomal modification techniques and anatural centromere is used to construct an artificial chromosome vector.In contrast, the bottom-up approach is an approach in which a portion ofa centromere sequence is obtained as a cloned DNA, which is thentransfected into a mammalian cell to construct a structure havingcentromere function. The artificial chromosome can stably replicate andcan be distributed as a chromosome independent from the nativechromosome of a host cell. The mouse-derived chromosome fragment is anyof chromosome fragments of mouse chromosomes 1 to 19, X, and Y (i.e., along-arm fragment formed by deleting at least 99.5% of all endogenousgenes from a long arm). This fragment includes a long-arm fragmentformed by deleting a long-arm distal region at a mouse chromosomelong-arm site proximal to the centromere, as defined above. Theconstruction of the artificial chromosome of the present invention isdescribed in Examples below, in particular Examples 1 to 5, and in FIGS.1 to 4. These Examples and figures illustrate how to construct anartificial chromosome from a fragment of mouse chromosome 11. The mouseartificial chromosome can also be constructed from a fragment of anotherchromosome in a substantially similar manner.

Sequence information of mouse chromosomes is available fromDDBJ/EMBL/GenBank or chromosome databases at Santa Cruz Biotechnology,Inc. and other organizations.

As used herein, the term “long arm” of a chromosome refers to achromosome region from the centromere side to the region containinggenes in a mouse chromosome. Meanwhile, the mouse chromosome has almostno short arm.

As used herein, the term “distal region” means a region distal from thecentromere (i.e., a region of the telomere side). On the other hand, the“proximal region” is referred to as a region near to the centromere(i.e., a region of the centromere side). The long-arm distal regionmeans a region being on the telomere side from a specific site of a longarm. The long-arm proximal region means a region being on the centromereside from a specific site of the long arm. This specific site is aposition at which at least 99.5%, preferably at least 99.7%, morepreferably 99.8%, and still more preferably 99.9 to 100% of allendogenous genes (or the number of all endogenous genes) that arepresent in the long arm of a chromosome derived from a mouse aredeleted.

As used herein, the term “retention rate” refers to a proportion ofcells having artificial chromosome in culture cells or tissue cells of amouse.

As used herein, the term “DNA sequence insertion site” means aninsertion site for a target DNA (including a gene) sequence, forexample, a recognition site for a site-specific recombinase. Examples ofsuch a recognition site include, but are not limited to, loxP (a Crerecombinase recognition site), FRT (a Flp recombinase recognition site),φC31 attB and φC31 attP (φC31 recombinase recognition sites), R4 attBand R4 attP (R4 recombinase recognition sites), TP901-1 attB and TP901-1attP (TP901-1 recombinase recognition sites), and Bxb1 attB and Bxb1attP (Bxb1 recombinase recognition sites).

As used herein, the term “site-specific recombinase” refers to an enzymethat induces a specific recombination with a target DNA sequence at therecognition site of the enzyme. Examples of the enzyme include Creintegrase (also referred to as Cre recombinase), φC31 integrase, R4integrase, TP901-1 integrase, and Bxb1 integrase.

As used herein, the term “telomere sequence” refers to a naturaltelomere sequence derived from same or different species, or anartificial telomere sequence. Here, the same species means the samespecies as the mouse from which a chromosome fragment of an artificialchromosome vector is derived, whereas the different species means amammal (including a human) other than the mouse. In addition, theartificial telomere sequence refers to a sequence having a telomerefunction and prepared by synthesis, such as a (TTAGGG)n sequence (where“n” means the number of repeats). Introducing a telomere sequence intoan artificial chromosome can be performed by telomere truncation (i.e.,substitution by a telomere sequence) as disclosed in WO 00/10383 forexample. The telomere truncation can be used to shorten a chromosomeduring construction of the artificial chromosome of the presentinvention.

As used herein, the term “exogenous gene” or “exogenous DNA” refers to agene or DNA of interest contained in a vector, wherein the gene or DNAis inserted into a gene insertion site of the vector. The term means agene or DNA or a sequence thereof that is originally absent in a celland that is to be expressed in the cell.

As used herein, the term “mammal” include, but are not limited to,primates such as human, monkey, and chimpanzee, rodents such as a mouse,rat, hamster, and guinea pig, and ungulates such as a cow, pig, sheep,and goat.

As used herein, the term “embryonic stem cell” or “ES cell” refers to asemi-immortalized pluripotent stem cell that is established from aninner cell mass of a blastocyst of a fertilized egg derived from amammal (M. J. Evans and M. H. Kaufman (1981) Nature 292, 154-156; J. A.Thomson et al. (1999) Science 282, 1145-1147; J. A. Thomson et al.(1995) Proc. Natl. Acad. Sci. USA 92, 7844-7848; J. A. Thomson et al.(1996) Biol. Reprod. 55, 254-259; J. A. Thomson and V. S. Marshall(1998) Curr. Top. Dev. Biol. 38, 133-165). The cell havingcharacteristics equivalent to ES cell, which is artificially induced byreprogramming of a somatic cell, is an “induced pluripotent stem cell”or “iPS cell” (K. Takahashi and S. Yamanaka (2006) Cell 126, 663-676; K.Takahashi et al. (2007) Cell 131, 861-872; J. Yu et al. (2007) Science318, 1917-1920).

Preparation and Use of Mouse Artificial Chromosome Vector:

Hereinafter, the preparation and use of the mouse artificial chromosomevector of the present invention will be described. Specifically, theprocedures are described in Examples 1 to 5 (FIGS. 1 to 4) below.

(1) Preparation of Mouse Artificial Chromosome Vector

The artificial chromosome vector of the present invention can beprepared in accordance with a method comprising the following steps (a)to (c):

(a) obtaining a cell having a mouse chromosome;

(b) deleting a long-arm distal region of the mouse chromosome so as notto include a large part (i.e., from 99.5% to 100%) of endogenous genes(or the number of endogenous genes); and

(c) inserting one or more DNA sequence insertion sites into a long-armproximal region. The order of the steps (b) and (c) may beinterchangeable.

Step (a):

In order to prepare the artificial chromosome vector according to thepresent invention, a cell having a mouse chromosome is first to beproduced. For example, a mouse embryonic fibroblast (mChr11-BSr), whichis a mouse fibroblast carrying a drug resistance gene (e.g., blasticidinS resistance gene (BSr))-labeled mouse chromosome, is subjected to cellinfusion with a mouse A9 (neo), which is a mouse A9 cell (ATCCVA20110-2209) having a neo gene (i.e., a G418-resistant gene). Next, themouse A9 hybrid cell having the drug resistance gene-labeled mousechromosome, i.e. the mouse A9× mouse embryonic fibroblast (neo;mChr11-BSr), is used to transfer the chromosome into a cell having ahigh homologous recombination rate, thereby preparing the cell having amouse chromosome. The mouse fibroblast is available based on proceduresdescribed in literatures. For example, the mouse fibroblast can beestablished from C57B6 mouse commercially available from CLEA Japan,Inc. Examples of the available cell having a high homologousrecombination rate can include chicken DT40 cell (Dieken et al., NatureGenetics, 12, 174-182, 1996). Furthermore, the above-described transfercan be carried out using known chromosome transfer techniques, such asmicrocell fusion (Koi et al., Jpn. J. Cancer Res., 80, 413-418, 1973).

Step (b):

In a cell having a single mouse-derived chromosome, a long-arm distalregion of the mouse chromosome is deleted. It is important to delete (orremove or cleave out) a large part of endogenous genes present in a longarm and then to construct an artificial chromosome having a mousecentromere. It is also important to determine a cleavage site in orderto delete a region containing at least 99.5%, preferably at least 99.7%,more preferably at least 99.8%, and still more preferably 99.9 to 100%of all endogenous genes present in the long arm. By doing so, a cell,tissue, or individual, which has the artificial chromosome and isderived from a mammal such as rodent (preferably mouse), can stablyretain the artificial chromosome at a high retention rate, and it can beused for precise analysis of a gene (a group of genes) of interest andfor production of materials. The above-described endogenous genes can bedeleted by telomere truncation as disclosed in, for example, WO00/10383. Specifically, a targeting vector having an artificial telomeresequence is constructed and is used to obtain a clone in which. a(artificial) telomere sequence has been inserted at a desired positionon the chromosome by homologous recombination in a cell having a mousechromosome. This makes it possible to obtain a deletion mutant viatelomere truncation. That is, the desired position (or site) is acleavage position of a long-arm distal region to be deleted. Theartificial telomere sequence is inserted into this position bysubstitution via the homologous recombination, so that the long-armdistal region is deleted. This position can be appropriately determineddepending on a target sequence design at the time of constructing atargeting vector. For example, in Examples below, a target sequence hasbeen designed based on the DNA sequence of AL671968 (GenBank AccessionNumber) on the long arm of mouse chromosome 11, so that the telomeretruncation occurs at a position of the telomere side from the targetsequence (see FIG. 9). As a results, a fragment of mouse chromosome 11having deletion of a large part of endogenous genes can be obtained. Forother chromosomes, the telomere truncation can be carried out similarly.

Step (c):

As a DNA sequence insertion site, a recognition site for a site-specificrecombinase can be preferably inserted. Specifically, the phenomenonthat a certain enzyme recognizes a specific recognition site, and causesDNA recombination specifically at the recognition site is known. Themouse artificial chromosome vector according to the present inventionuses a system having such an enzyme and its recognition site to insertor carry a gene or DNA sequence of interest. Examples of such a systeminclude a system having bacteriophage P1-derived Cre enzyme and itsrecognition site, i.e. loxP sequence (a Cre/loxP system; B. Sauer inMethods of Enzymology, 1993, 225, 890-900), a system having buddingyeast-derived Flp enzyme and its recognition site, i.e. FRT (FlpRecombination Target) sequence (a Flp/FRT system), a system havingStreptomyces phage-derived φC31 integrase and its recognition site, i.e.φC31 attB/attP sequences, a system having R4 integrase and itsrecognition site, i.e. R4 attB/attP sequences, a system having TP901-1integrase and its recognition site, TP901-1 attB/attP sequences, and asystem having Bxb1 integrase and its recognition site, i.e. Bxb1attB/attP sequences. As long as functioning as a DNA sequence insertionsite, the system is not limited to the above systems.

In order to insert a recognition site for such a site-specificrecombinase, known methods, such as homologous recombination, can beemployed. The position and number of insertion can be appropriatelydetermined in a long-arm proximal region and a short-arm proximalregion.

According to the present invention, one of certain recognition sites ordifferent recognition sites can be inserted. The design of a recognitionsite enables specifying an insertion site for an exogenous gene orexogenous DNA, so that the insertion site is fixed and no unexpectedpositional effects are thus exerted. A mouse artificial chromosome asillustrated in Examples below can express, in a tissue-specific fashion,a gene inserted at a loxP sequence that is inserted into BX572640 locuson mouse chromosome 11 and that is a recognition site for a sitespecific recombinase (FIGS. 11, 20, and 24).

Preferably, the mouse artificial chromosome vector having a DNA sequenceinsertion site according to the present invention may beforehand have areporter gene while preserving an insertion site for a target gene orDNA sequence. Examples of the reporter gene include, but are not limitedto, fluorescent protein genes (e.g., green fluorescent protein (GFP orEGFP) gene, yellow fluorescent protein (YFP) gene), atag-protein-encoding DNA, β-galactosidase gene, and luciferase gene.Preferred is GFP or EGFP.

The mouse artificial chromosome vector according to the presentinvention may further comprise a selection marker gene. The selectionmarker is effective in selecting a cell transformed by the vector. Theselection marker gene is represented by either a positive selectionmarker gene or a negative selection marker gene, or both. Examples ofthe positive selection marker gene include drug resistance genes such asa neomycin-resistant gene, an ampicillin-resistant gene, a blasticidin S(BS)-resistant gene, a puromycin-resistant gene, a geneticin(G418)-resistant gene, and a hygromycin-resistant gene. In addition,examples of the negative selection marker gene include a herpes simplexthymidine kinase (HSV-TK) gene, and a diphtheria toxin A fragment (DT-A)gene. In general, the HSV-TK is used in combination with ganciclovir orciclovir.

Homologous recombination can be preferably used as a technique forinserting a reporter gene or a target exogenous gene or DNA into themouse artificial chromosome vector according to the present invention.The homologous recombination can be carried out using a targeting vectorwhich is obtained by ligating an DNA cassette to be inserted betweensequences (5′ arm and 3′ arm) homologous to nucleotide sequences of 5′and 3′ regions (each having approximately 1 to 4 kb, preferablyapproximately 2 to 4 kb) at an insertion position of the mousechromosome. Examples of the vector that can be used for this purposeinclude a plasmid, a phage, a cosmid, and a virus. Preferred is aplasmid. Examples of a basic plasmid for targeting vector constructioninclude, but are not limited to, V907 and V913 (Lexicon Genetics). Thebasic vector may contain one or two or more sequences or elements thatare generally inserted for the vector construction, such as a promoter,an enhancer, a selection marker gene, a replication origin, and thelike.

The mouse artificial chromosome prepared using the above procedurescomprises a mouse-derived chromosome fragment (which comprises a naturalcentromere, a long-arm fragment formed by deleting at least 99% orpreferably at least 99.5% of endogenous genes, and a short arm (ifpresent)), and an artificial telomere sequence. The above centromerealso consists of an entire mouse chromosome centromere structure whichis used for the artificial chromosome construction.

An example of the mouse artificial chromosome vector of the presentinvention is the mouse artificial chromosome vector as prepared inExamples below. This artificial chromosome is a vector produced bydeleting a long-arm distal region of mouse chromosome 11 at AL671968(FIGS. 1, 3, 4, 9, and 10). This vector comprises, as a basic structure,the mouse artificial chromosome contained in the deposited cell lineDT40 B6bT-1 (FERM BP-11128), which vector is herein designated as DT40(MAC). Because this vector has a basic structure, this DNA structure cancomprise the following insertions such as a DNA sequence insertion site,a selection marker gene, and an exogenous gene (or DNA).

The above mouse artificial chromosome vector preferably comprises one ormore DNA sequence insertion sites such as a recognition site for asite-specific recombinase (e.g., a loxP sequence which is a Cre enzymerecognition site) (FIGS. 1, 3, 4, 11, 20, and 24). Examples of therecognition site for the site-specific recombinase include, but are notlimited to, loxP sequences of GFP-PGKneo-loxP-3′ HPRT type, 5′HPRT-loxP-hyg type, PGKneo-loxP-3′ HPRT type, or GFP-5′ HPRT-loxP-PGKhygtype, wherein the GFP represents a green fluorescent protein gene, thePGKneo represents a phosphoglycerate kinase promoter/neomycin-resistantgene cassette, the HPRT represents a hypoxanthine guaninephosphoribosyltransferase gene, and the hyg represents ahygromycin-resistant gene.

The above-described mouse artificial chromosome vector may furthercomprise a reporter gene or a selection marker gene (e.g., a positiveselection marker gene, or a negative selection marker gene). The vectormay further comprise a target exogenous gene or DNA sequence.

The advantages of the mouse artificial chromosome vector according tothe present invention include advantages of conventional artificialchromosome vectors as follows: 1) The vector is independently maintainedwithout being inserted into a host chromosome, so that no disruption ofa host gene is caused; 2) The vector is stably retained at a certaincopy number (which may be a plurality of (or multiple) copies) and isexposed to the physiological expression regulation of a host cell, sothat the overexpression or loss of expression of the inserted gene isnot caused; 3) DNA that can be introduced has no size limitation and soit becomes possible to introduce a gene comprising expression regulatoryregion or a plurality of genes/isoforms. In addition, the retention rateof the vector in a rodent cell or individual increases compared withthat of conventional artificial chromosomes, and a transgene can bestably expressed for a long term and the rate of genetic transmission ofthe vector to offspring is improved, thereby increasing the efficiencyof producing a transgenic mouse. Further, 4) there is less variationamong tissues after introduction of the vector, that is, the retentionrate is 90% or higher in any tissue. Even a hematopoietic tissue, whichusually has a retention rate of less than 20% in the case of the HAC,has a retention rate of 90% or higher.

(2) Introduction of Exogenous Gene or DNA

An exogenous gene or DNA can be introduced into the mouse artificialchromosome vector according to the present invention.

The size of the exogenous gene or DNA sequence is not particularlylimited, but may be 20 kb or less or may exceed 20 kb, such as 50 kb ormore, 100 kb or more, 200 kb or more, 500 kb or more, 700 kb or more, 1Mb or more, 10 Mb or more, 20 Mb or more, 30 Mb or more, 40 Mb or more,and 50 Mb or more. The vector of the present invention can carry anexogenous DNA (chromosome fragment) of 1 Mb or more as seen in HACvector, the size of which is difficult to be carried by artificialchromosome vectors such as BAC, PAC, and YAC vectors. Moreover, thevector of the present invention can stably retain a large-size exogenousgene or DNA of 200 kb or more, for example 1 Mb or more, at a higherretention rate (90% or more) than the HAC vector in a mammalian cell ortissue or in a non-human animal individual, preferably in a rodent cell,tissue or individual.

An embodiment of the present invention provides a vector and a methodfor preparing the same, wherein the vector is able to stably maintain abig-size exogenous gene or DNA of 200 kb or more in a rodent cell orindividual at a retention rate of 90% or more.

The exogenous gene or DNA refers to a nucleic acid sequence that isintroduced from outside of a cell of interest, but is not particularlylimited. This gene or DNA may be derived from any organism, tissue orcell, preferably from a mammal, and more preferably from a human.Examples of such a gene or DNA include, but are not limited to, genes orDNAs encoding polypeptides such as cytokines, hormones, growth factors,neurotrophic factors, hematopoietic factors, immunoglobulins, Gprotein-coupled receptors, and enzymes, in addition to genes or DNAsused for treatment involved in various diseases such as tumor, musculardystrophy, hemophilia, neurodegenerative diseases (e.g., Alzheimer'sdisease, Huntington's disease, and Parkinson's disease), autoimmunediseases, allergic diseases, and genetic diseases, genes (gene groups)or DNAs encoding (human) drug-metabolizing enzymes, (human) drugmetabolism-related genes, long-arm or short-arm DNAs of humanchromosomes, and (human) genomic libraries.

Examples of the cytokines include interferons (e.g., IF-α, IF-β, andIF-γ), interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-11, and IL-12),tumor necrosis factors (e.g., TNF-α, and TNF-β), and TGF-β familyproteins (e.g., bone morphogenic protein (BMP)).

Examples of the hormones include growth hormones, human chorionicgonadotropin (hCG), human placental lactogen (hPL), human pituitarygonadotropic hormone, thyroid-stimulating hormone (TSH), luteinizinghormone-releasing factor, insulin, glucagon, somatostatin, andprolactin.

Examples of the growth factors or neurotrophic factors includeinsulin-like growth factor, brain-derived neurotrophic factor (BDNF),albumin-fused ciliary neurotrophic factor, platelet-derived neurotrophicfactor (PDNF), transforming growth factor, nerve growth factor (NGF),and TNF growth factor.

Examples of the coagulation or hemolysis factors include Factor VII,Factor VIII, Factor X, and t-PA.

Examples of the hematopoietic factors include erythropoietin,(granulocyte) colony-stimulating factor, and thrombopoietin.

Examples of the immunoglobulins include human antibodies, humanizedantibodies, chimeric antibodies, and recombinant antibodies such assynthetic antibodies, which are elicited against various antigens.

Examples of the G protein-coupled receptors include adrenergic receptor,muscarinic acetylcholine receptor, adenosine receptor, GABA receptor(type B), angiotensin receptor, cholecystokinin receptor, dopaminereceptor, glucagon receptor, histamine receptor, odorant receptor,opioid receptor, secretin receptor, somatostatin receptor, gastrinreceptor, and P2Y receptor.

Examples of the enzymes include asparaginase, superoxide dismutase,uricase, streptokinase, dopamine synthase, and adenosine deaminase.

Examples of the genes used for treatment involved in various diseasessuch as tumor, muscular dystrophy, neurodegenerative diseases (e.g.,Alzheimer's disease, Huntington's disease, and Parkinson's disease),autoimmune diseases, allergic diseases, and genetic diseases includedystrophin gene, IL-12 gene, TNF-α gene, tumor suppressor genes,dopamine synthase gene, and genes encoding genetically deficientenzymes.

The drug-metabolizing enzymes are involved in the metabolic reactions todegrade or excrete xenobiotics such as drugs or toxins. Examples of theenzymes include enzymes involved in the phase I reaction (e.g.,oxidation, reduction, and hydrolysis) or the phase II reaction(conjugation). Examples of the enzymes involved in the phase I reactioninclude known enzymes such as cytochrome P450 (“CYP”), specifically,CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A, CYP4A,CYP4B, and subfamilies thereof, and CESs. With regard to the CYPsubfamily, examples of the CYP3A subfamily include CYP3A4, CYP3A43,CYP3A5, and CYP3A7. In addition, examples of the CYP2C subfamily includeCYP2C8, CYP2C9, CYP2C18, and CYP2C19. For a reference, CYP3A-MAC asdescribed in Examples below means a CYP3A cluster, which includesCYP3A4, CYP3A43, CYP3A5, and CYP3A7. On the other hand, examples of theenzymes involved in the phase II reaction (conjugation) include UGT1 andUGT2.

Examples of the drug metabolism-related genes include genes encodingtransporters and genes encoding nuclear receptors. Examples of the genesencoding transporters include MDR1, MDR2, MRP2, OAT, OATP, OCT, andBCRP. Examples of the genes encoding nuclear receptors include PXR, AhR,CAR, and PPARα.

In view of the above, a drug-metabolism-related exogenous DNA sequencethat is capable of introduced into the vector of the present inventioncan comprise at least one gene sequence or at least two gene sequencesselected from the group consisting of genes encoding enzymes involved inthe phase I reaction, genes encoding enzymes involved in the phase IIreaction, genes encoding transporters, and genes encoding nuclearreceptors.

At least one insulator sequence may be present at a proximal region orboth sides of the insertion site of an exogenous gene or DNA containedin the mouse artificial chromosome vector of the present invention. Theinsulator sequence exerts an enhancer blocking effect (i.e., whereinadjacent genes do not affect each other) or a chromosome boundary effect(i.e., wherein a region promoting gene expression is separated anddistinguished from a region inhibiting the gene expression). Examples ofsuch a sequence include human β-globin HS1 to HS5 and chicken β-globinHS4.

The exogenous gene or DNA can be introduced by using the above-describedsite-specific recombinase system which is inserted as theabove-described DNA sequence insertion site. For example, a targetingvector is constructed that comprises an exogenous gene or DNA and a loxPsequence which is the recognition site for Cre enzyme, or a chromosomefragment is constructed that comprises an exogenous gene or DNA and aninserted loxP sequence which is the recognition site for Cre enzyme.Then, the Cre enzyme is expressed in a cell having the mouse artificialchromosome vector according to the present invention, thereby inducing asite-specific recombination at the loxP sequence with the targetingvector or the chromosome fragment to introduce the exogenous gene orDNA.

The mouse artificial chromosome vector according to the presentinvention can incorporate a circular DNA having a recognition site(e.g., a loxP sequence or an FRT sequence) for a site-specificrecombinase. The DNA may be inserted that has been cloned by using knownvectors such as plasmids for host Escherichia coli and circular YAC forhost yeast. Preferable loxP sequence is a wild type sequence derivedfrom P1 phage, and the insertion reaction of the circular insert intothe loxP sequence on the artificial chromosome vector using the Creenzyme is reversible. Once the circular insert is inserted, two loxPsequences are left on the artificial chromosome vector. Because of this,reexpression of the Cre enzyme may cause a reversible reaction thatcleaves out the circular insert, and thus it becomes difficult tofurther modify the artificial chromosome vector so as to secondarilyinsert the insert. When a mutant loxP sequence with nucleotidesubstitution or a combination of attB/attP sequences which is therecognition site for φC31 integrase is used, the reversible reactiondoes not occur and so it becomes possible to construct a system intowhich a plurality of circular inserts are sequentially inserted.

(3) Transfer of Mouse Artificial Chromosome Vector into Cell andCreation of Non-human Animal

The mouse artificial chromosome vector of the present invention, or themouse artificial chromosome vector comprising an exogenous gene or DNAaccording to the present invention, can be transferred or introducedinto any cell. Examples of the method to achieve that goal includemicrocell fusion, lipofection, a calcium phosphate method,microinjection, and electroporation. Preferred method is the microcellfusion.

The microcell fusion is a method for transferring a mouse artificialchromosome vector into a desirable cell by microcell fusion between afirst cell (e.g., mouse A9 cell) which has an ability to formmicronuclei and contains the mouse artificial chromosome vector of thepresent invention and a second desirable cell. The first cell having anability to form micronuclei is treated with a polyploid inducer (e.g.,colcemid or colchicine) to form a multinucleated cell havingmicronuclei. Then, the cell is subjected to cytochalasin treatment toform microcells, which are subsequently fused with a desirable cell.

Examples of the cells capable of introducing the above vector includeanimal cells, preferably mammalian cells including human cells, such asgermline cells (e.g., oocyte and spermatocyte), stem cells (e.g.,embryonic stem (ES) cells, germline stem (GS) cells, somatic stemcells), somatic cells, embryonal cells, adult cells, normal cells,disease cells, primary cultured cells, subcultured cells, and celllines. Examples of the stem cells include pluripotent stem cells (e.g.,ES cells, embryonic germline (EG) cells, embryonic carcinoma (EC) cells,mGS cells, and human mesenchymal stem cells), induced pluripotent stem(iPS) cells, and nuclear transfer clone embryo-derived embryonic stem(ntES) cells. The preferred cells can be selected from the groupconsisting of mammalian (preferably a rodent including mouse) somaticcells, non-human germline cells, stem cells, and precursor cells. Whenthe cell is derived from a mammal such as a rodent and the vector of thepresent invention is introduced into the cell or tissue of the mammal(e.g., a rodent such as mouse), the vector can be more stably retained.That is, drop-out of the vector from the cell significantly decreases,or the drop-out never happens.

Examples of the cells include hepatocyte, enterocyte, renal cell,splenocyte, lung cell, cardiac cell, skeletal muscle cell, brain cell,bone marrow cell, lymphocyte, megakaryocyte, spermatocyte, and oocyte.

Examples of the tissues include liver, intestine, kidney, spleen, lung,heart, skeletal muscle, brain, bone marrow, testis, and ovary.

ES cells can be established and maintained as follows: first, an innercell mass is removed from the blastocyst of a fertilized egg of ananimal of interest; the inner cell mass is then cultured using amitomycin C-treated mouse embryonic fibroblast as a feeder, therebyestablishing the ES cells which are then maintained (M. J. Evans and M.H. Kaufman (1981) Nature 292, 154-156).

iPS cells are generated by introducing specific reprogramming factors(DNAs or proteins) into a somatic cell (including a somatic stem cell),by culturing and subculturing the cell in appropriate media, therebyproducing colonies after about 3 to 5 weeks. Examples of the knowncombination of reprogramming factors include a combination of Oct3/4,Sox2, Klf4, and c-Myc; a combination of Oct3/4, Sox2, and Klf4; acombination of Oct4, Sox2, Nanog, and Lin28; and a combination ofOct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28 (K. Takahashi and S.Yamanaka, Cell 126, 663-676 (2006); WO 2007/069666; M. Nakagawa et al.,Nat. Biotechnol. 26, 101-106 (2008); K. Takahashi et al., Cell 131,861-872 (2007); J. Yu et al., Science 318, 1917-1920 (2007); J. Liao etal., Cell Res. 18, 600-603 (2008)). Examples of the culture include:using a mitomycin C-treated mouse embryonic fibroblast cell line (e.g.,STO) as a feeder cell; and culturing, at about 37° C., a somatic cell(approximately 10⁴ to 10⁵ cells/cm²) with introduced vector on thefeeder cell layer using a medium for ES cells. The feeder cell is notnecessarily required (Takahashi, K. et al., Cell 131, 861-872 (2007)).Examples of the basic medium include Dulbecco's Modified Eagle's Medium(DMEM), Ham's F-12 medium, and a mixture thereof. Examples of theES-cell medium that can be used include a medium for mouse ES cells anda medium for primate ES cells (Reprocell Inc.).

ES cells and iPS cells are known to contribute to the germlinetransmission. Hence, a non-human animal (or a transgenic animal(excluding a human)) can be generated by a method comprising:introducing into the ES cell or iPS cell, the mouse artificialchromosome vector having a gene or DNA of interest according to thepresent invention; injecting the cell into the blastocyst of an embryoderived from the same mammalian species as that from which the cell isderived; and transplanting the embryo into the uterus of a fostermother, which gives birth to an animal(s). Homozygous animals arecreated by further crossing between a male and a female of the resultingtransgenic animals. In addition, their offspring can also be produced.

An exogenous gene or DNA, such as a human antibody gene, a gene fortreating a disease, and a drug metabolism-related gene, is introducedinto pluripotent cells such as ES cells and iPS cells or the above othercells via the mouse artificial chromosome vector of the presentinvention, thereby producing a cell or non-human animal that can producea human antibody. In addition, a cell that can produce a therapeuticprotein can be generated. Furthermore, a non-human animal model for adisease such as a drug metabolism-related disease can also be generated.

In some cases, it is preferable that such a non-human animal has adisrupted endogenous gene or decreased expression of the endogenous genecorresponding to the exogenous gene included in the mouse artificialchromosome vector. Gene targeting can be employed as the disruptiontechnique. RNAi method can be used as a method for decreasing endogenousgene expression. Examples of such an exogenous gene include a drugmetabolism-related gene and a human antibody gene. A non-human animalwhose endogenous gene has been disrupted can be created by crossingbetween a non-human chimeric animal having the mouse artificialchromosome vector containing the exogenous gene or its offspring and achimeric animal having a deletion of the entire cluster of thecorresponding endogenous gene or its offspring to yield an animal havinga heterozygous deletion of the endogenous gene, and by further crossingbetween the heterozygous animals.

The cell or transgenic non-human animal having a mouse artificialchromosome vector can be produced by the above-described techniques.Examples of the specific non-human animal include a rodent, such asmouse or rat, having the mouse artificial chromosome vector.

Thus, the present invention provides a cell or non-human animalcomprising the mouse artificial chromosome vector.

Furthermore, the cell, tissue, or organ obtained from the non-humananimal of the present invention can be used to generate a cell line thatproduces a protein expressed by the exogenous gene.

(4) Method for Producing Useful Protein

The present invention provides a method for producing a protein,comprising: culturing a cell comprising a mouse artificial chromosomevector comprising a sequence of an exogenous DNA in a situation capableof expression; and collecting a protein encoded by the DNA.

Examples of the protein include the above therapeutically,agriculturally, or industrially useful proteins and polypeptides. DNAsencoding these proteins or polypeptides are each inserted into the mouseartificial chromosome vector so that the DNA can be expressed in thepresence of a promoter (and an enhancer if needed). Then, appropriatecells are transformed or transfected with the DNA. The resulting cellsare cultured, and the DNA is expressed to produce the protein orpeptide. After that, the protein or peptide is collected from the cellsor medium.

Examples of the cells that can be used include eukaryotic cells, e.g.,insect cells such as an Sf cell, bird cells, yeasts, and plant cells, inaddition to mammalian cells.

Culture conditions including a medium are selected depending on celltypes, and can employ known conditions as culture conditions. Examplesof the media for animal cells include MEM medium, DMEM medium, Ham's F12medium, Eagle's MEM medium, Iscove's EME medium, RPMI1640 medium, and amixture thereof.

Collecting (or isolating) a protein or polypeptide can be performed byusing chromatography techniques, such as gel filtration chromatography,ion exchange chromatography, affinity chromatography, chromatography,HPLC, and FPLC, salting-out, ammonium sulfate precipitation, organicsolvent precipitation, ultrafiltration, and crystallization, alone or incombination.

(5) Method for Producing Human Antibody

The present invention further provides a method for producing a humanantibody, comprising: using the above non-human animal carrying themouse artificial chromosome vector comprising a human antibody gene toproduce the human antibody; and collecting the human antibody.

Examples of genes encoding a human antibody include genes encoding anyclass of human IgG, IgM, IgA, IgD, and IgE or any subclass of humanIgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The preferred human antibodygene is a gene encoding an IgG class or a subclass thereof.

A human antibody consists of two heavy (H) chains having an identicalsequence and two light (L) chains having an identical sequence. Both Hand L chains comprise a variable region and a constant region. Thevariable region of the human H or L chain has three complementaritydetermining regions (CDR1, CDR2, and CDR3 in the order from N-terminalside to C-terminal side) and four framework regions (FR1, FR2, FR3, andFR4 in the order from N-terminal side to C-terminal side). Threeindividual CDR sequences of the human H or L chain determine thespecificity of an antibody.

A human IgG antibody comprises a μ chain of the heavy chain and a X or Kchain of the light chain. These antibody chain genes are present onhuman chromosome 14, chromosome 22, and chromosome 2, respectively. As ahuman antibody gene used in the present invention, a human chromosomefragment containing each antibody gene locus is used to integrate eachlocus into the same or different mouse artificial chromosome. Antibodygene sequences are available from databases of NCBI (U.S.). This seriesof procedures are an improved version of the techniques disclosed inJapanese Patent Publication (Kokai) NO. 2005-230020A.

A non-human animal that can produce an intact human antibody can beproduced by crossing between a non-human animal comprising a mouseartificial chromosome vector comprising a human μ-chain gene locus andthe same non-human animal species comprising a mouse artificialchromosome vector comprising a human λ- and/or κ-chain gene loci togenerate a non-human chimeric animal having both H- and L-chain geneloci and offspring thereof.

The resulting non-human animal (e.g., a rodent such as mouse) that canproduce an intact human antibody is immunized with a specific antigenicpeptide or polypeptide. Then, the human antibody is isolated from bloodof the animal. Through the above process, the human antibody can beproduced.

Alternatively, a non-human animal is immunized with a specific antigen.Next, the spleen of the non-human animal is removed. Then, cells fromthe spleen can be made to be fused with myeloma cells to yieldhybridomas producing a monoclonal antibody.

(6) Method for Screening for Therapeutic Substance

The present invention further provides a method for screening for asubstance effective in treating a disease, comprising: administering acandidate drug to a disease-model animal of the above non-human animal;and evaluating a therapeutic effect of the dug.

The disease-model non-human animal is an artificially produced animalhaving a disease caused by abnormalities such as abnormal biologicalfunctions due to deficiency, mutation or the like of a certain protein,abnormal drug metabolism, and abnormal chromosomes. Examples of thenon-human animal model having an abnormal chromosome include, but arenot limited to, animals having trisomy of human chromosome 18 or 21.

These non-human animals can be created by the method comprising:constructing a gene or a chromosome fragment having the aboveabnormality; incorporating the gene or chromosome fragment into themouse artificial chromosome vector of the present invention; introducingthe vector into ES cells or iPS cells; injecting the obtained cells intothe blastocyst of a fertilized egg; transplanting the egg into theuterus of a foster mother; and delivering offspring.

A substance effective in treating the above disease can be screened byadministering a candidate drug to a non-human animal created above andthen evaluating a therapeutic effect of the drug.

Examples of the candidate drug include, but are not limited to,low-molecular-weight compounds, polymers, (glyco)proteins, peptides,(phospho or glyco)lipids, and sugars.

(7) Method for Testing Pharmacological Effect, Metabolism, or Toxicityof Drug or Food

In an embodiment of the present invention, the invention also provides amethod for testing a pharmacological effect and/or metabolism and/ortoxicity of a drug or food, comprising: administering a drug or food tothe above non-human animal or a cell, organ, or tissue thereof, whereinthe animal, cell, organ, or tissue comprises the mouse artificialchromosome vector comprising a human drug metabolism-related gene; anddetermining a pharmacological effect and/or metabolism and/or toxicityof the drug or food.

The present invention further provides a method for testing toxicity ofa drug or food, comprising: coculturing a drug and/or food and a culturecell or bacterium with a microsome or microsome fraction S9 as obtainedfrom the above non-human animal, wherein the animal comprises the mouseartificial chromosome vector comprising a human drug-metabolism-relatedgene; and determining an (adverse) influence (e.g., a mutation) on theculture cell or bacterium by the drug or food.

The human drug metabolism-related gene is as exemplified above. Also, amethod for creating a non-human animal is as described above.

In the above-described method using the above non-human animalcomprising the mouse artificial chromosome vector comprising a humandrug metabolism-related gene, for example, the animal's conditions areobserved and influences on organs or chromosomes are tested, therebybeing able to determine a pharmacological effect, metabolism, ortoxicity of the drug or food.

In another method of the present invention, a microsome or microsomefraction S9 (i.e., the 9000×g fraction containing a large number ofenzymes that catalyze hydrolysis, reduction, oxidation, or conjugation)as obtained from a non-human animal is cocultured with a culture cell(in particular, animal cell, preferably mammalian cell) or bacterialcell (preferably salmonella) in the presence of a drug and/or food.Toxicity of the drug or food on the cells can be detected by Ames testor a micronucleus test. The Ames test determines the toxicity based onmutations of salmonella. The micronucleus test determines the toxicitybased on abnormalities of chromosomes in a cell nucleus. These testshave been well known, and can be used in the methods according to thepresent invention.

Hereinafter, the present invention will further be described in moredetail by referring to Examples. The scope of the present invention,however, is not limited to the specific Examples.

EXAMPLES Example 1 Construction of the Mouse Artificial ChromosomeVector MAC

By performing telomere truncation of a mouse chromosome, mouseartificial chromosome MAC [DT40 (B6bT)] containing no endogenous gene isconstructed (FIG. 1).

[A] Establishment of Hybrid Cells of A9 Cells and Mouse Fibroblast (Neo;mChr11-BSr)

Mouse embryonic fibroblast (mChr11-BSr), which is mouse fibroblastcontaining mouse chromosome 11 labeled with drug resistant gene (Bsrgene), is cell-fused with mouse A9 (neo) obtained by inserting neo gene,i.e., G418 resistant gene, into known mouse A9 cells to establish mouseA9× mouse embryonic fibroblast hybrid (neo; mChr11-BSr), i.e., mouse A9hybrid cell retaining a mouse chromosome labeled with a drug resistantgene. To introduce the mouse chromosome labeled with a drug resistantgene into chicken DT40 cells having high homologous recombinationfrequency by microcell fusion, a mouse chromosome labeled with a drugresistant gene is introduced into mouse A9 cell known to have a highmicrocell formation rate.

[A. 1] Cell Fusion and Isolation of Dual Drug Resistant Clone

Cell surfaces of mouse embryonic fibroblast (mChr11-BSr), which is mousefibroblast established from C57B6 lineage-based mouse embryo availablefrom CLEA Japan, Inc. and in which a drug resistant gene (Bsr gene) isinserted into the mouse chromosome and mouse A9 (neo), which is mouse A9cell in which neo gene (i.e., G418 resistant gene) is inserted, arewashed with PBS (−) separately. Cells are then dispersed by addingtrypsin, suspended in culture medium (10% FBS, DMEM), and 1×10⁶ cells ofsaid cells are simultaneously inoculated into a culture flask (25 cm²)and cultured for 1 day. The cell surfaces were washed twice with PBS(−), treated with 3 ml of PEG (1:1.4) solution [5 g, PEG1000, cat:165-09085, Wako, is dissolved in 6 ml of serum free DMEM, and 1 ml ofdimethyl sulfoxide was added thereto, and the mixture is sterilized byfiltration] for 1 min, and further treated with 3 ml of PEG (1:3)solution [5 g, PEG1000, cat: 165-09085, Wako, is dissolved in 15 ml ofserum free DMEM, and sterilized by filtration] for 1 min. The PEGsolution was aspirated off, and the cells were washed three times withserum free DMEM, and cultured for 1 day with common culture medium (10%FBS, DMEM). Cell surfaces were washed with PBS (−) and the cells weredispersed by adding trypsin, and after suspended in a double selectionculture medium containing G418 (800 μg/ml) and blasticidin S (4 μg/ml),inoculated into plastic culture dishes and then subjected to selectionculture for 2 to 3 weeks. Total three resistant colonies obtained by twocell fusions were isolated, amplified, and subjected to the followinganalysis (clone name: mouse A9× mouse embryonic fibroblast hybrid (neo;mChr11-BSr)).

[A. 2] Selection of Hybrid Cells

[A. 2. 1] PCR

From the dual drug resistant clone, genomic DNA was extracted and PCRwas carried out by using the following primers to confirm that the mousechromosome labeled with the drug resistant gene (Bsr gene) is retained.

Bsr R1: (SEQ ID NO: 1) 5′ CATGTGGGAGCGGCAATTC 3′ Bsr L1: (SEQ ID NO: 2)5′ TTGAGTGGAATGAGTTCTTCAATCG 3′

For PCR, GeneAmp 9600 (manufactured by PerkinElmer, Inc.) was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) used werethose included in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 95° C. for 10min, 35 cycles of 94° C. for 30 sec, 60° C. for 30 sec, and 72° C. for30 sec were carried out. As a result of PCR, all clones out of the threeclones were found to be positive.

TABLE 1 Mouse A9 x mouse Mouse embryonic fibroblast embryonic hybridfibroblast (neo, mChr11-BSr) (mChr11-BSr) 5 6 7 1 2 A9(neo) Bsr R1/L1 ◯◯ ◯ ◯ ◯ X[A. 2. 2] Quinacrine-Hoechst Double Staining

Clones found to be positive by the above PCR analysis were subjected toQuinacrine-Hoechst double staining For Quinacrine-Hoechst doublestaining, a chromosome slide was first immersed in 50 ml of McIlvainesolution [11.18 g of citric acid monohydrate and 13.29 g of disodiumhydrogen phosphate are dissolved in 1 L of water and autoclaved], andthen immersed for 20 min in 50 ml of McIlvaine solution in whichQuinacrine [cat: Q2876, SIGMA] is dissolved at 60 μg/ml, and thebackside of the chromosome slide was washed with tap water, immersed inMcIlvaine solution, and then immersed for 15 min in 50 ml of McIlvainesolution in which Hoechst [cat: B-2883] is dissolved at 0.5 μg/ml, andthe slide was covered with a cover glass. As a result of fluorescentmicroscopic observation, most nuclear types generally having 2n becameto have 4n or more in all three clones. It was particularly found that,in clone A9 (21-B6b) 7, the mouse fibroblast retaining the mousechromosome labeled with the drug resistant gene (Bsr gene) and mouse A9cells into which neo gene (i.e., G418 resistant gene) was inserted, werecell-fused in one to one ratio (FIG. 6).

TABLE 2 Metaphase 2n 4n 8n Total Mouse A9 x mouse embryonic fibroblast 110 9 20 hybrid (neo, mChr11-BSr)-5 Mouse A9 x mouse embryonic fibroblast3 2 15 20 hybrid (neo, mChr11-BSr)-6 Mouse A9 x mouse embryonicfibroblast 5 14 1 20 hybrid (neo, mChr11-BSr)-7 A9(neo) 18 2 20

From these results, it was concluded that mouse A9× mouse embryonicfibroblast hybrid (neo; mChr11-BSr) had a labeled mouse chromosome.

[B] Introduction of Mouse Chromosome Labeled with Drug Resistant Geneinto DT40 Cells

A mouse chromosome labeled with a drug resistant gene from mouse A9×mouse embryonic fibroblast hybrid (neo; mChr11-BSr), which is a mouse A9hybrid cell containing a mouse chromosome labeled with a drug resistantgene, was introduced into DT40, which is a chicken DT40 cell. To performefficiently the insertion of the loxP sequence as a DNA sequenceinsertion site to a mouse chromosome by identification of mousechromosome number, telomere truncation (i.e., site specific cleavage ofa chromosome via insertion of an artificial telomere), and homologousrecombination, a mouse chromosome labeled with a drug resistant gene isintroduced into DT40, which is a chicken DT40 cell having a highhomologous recombination frequency, by microcell fusion.

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

For efficient identification of chromosome and modification ofchromosome, the mouse chromosome was transferred from A9× mouseembryonic fibroblast hybrid (neo; mChr11-BSr) 7, which is a A9 hybridcell clone, to DT40 which is a chicken DT40 cell having a highhomologous recombination frequency. When the A9× mouse embryonicfibroblast hybrid (neo; mChr11-BSr) 7, which is a donor cell cultured inflask×24, reached 70% confluency in each flask, colcemid treatment(colcemid 0.05 μg/ml, 20% FCS, DMEM) was carried out for 48 hours at 37°C. under 5% CO₂ condition. Upon the completion of the colcemidtreatment, the medium inside the flask was aspirated off, and the flaskwas filled up to 90% with cytochalasin B. The flask was placed in acontainer dedicated to a large size high-speed centrifuge (BECKMAN) andwarm water (34° C.) was added to the level at which the flask was stillnot covered, and centrifugation was carried out (Rortor ID10.500, 8,000rpm, 1 h, 34° C.). Upon the completion of the centrifugation,cytochalasin B was recovered, and pellets in each flask were collectedin 15 ml tube each containing 2 ml of serum free culture medium DMEM.After slow filtration in the order of 8 μm→5 μm→3 μm filters, each tubewas centrifuged (1,200 rpm, 5 min at R.T). The supernatant was aspiratedoff, and pellets from each tube were combined, recovered and suspendedin 5 ml of serum free culture medium DMEM, and centrifuged (2000 rpm 5min).

Since DT40 cells as a recipient are floating cells, they need to be inadherent state once. In order to adhere DT40 on one well of 6-well plate(Nunc), one well was incubated overnight at 37° C. with 1.5 ml ofpoly-L-lysine (SIGMA), which has been adjusted to 50 μg/ml, for coating.The poly-L-lysine was recovered, and the plate was washed with PBS (−),and approximately 1×10⁷ DT40 cells were gently plated onto 2 ml of serumfree culture medium (DMEM). The plate itself was set in a centrifuge(Beckman) and centrifuged at 37° C., 1200 rpm, for 3 min to obtainadhered DT40.

Purified microcells were suspended in 2 ml of serum free culture mediumcontaining PHA-P (SIGMA), and gently plated onto the adhered DT40 fromwhich the serum free culture medium (DMEM) were depleted. The plate wascentrifuged for 3 min at 37° C., 1200 rpm. The supernatant was removedand fused exactly for 1 min with 1 ml of PEG1000 (Wako) solution [5 g ofPEG1000 is completely dissolved in serum free DMEM culture medium, addedwith 1 ml of dimethyl sulfoxide, and sterilized by filtration]. Thecells were washed four times with 4 ml of serum free culture medium(DMEM), and by pipetting with 3 ml of common culture medium for DT40,the adhered DT40 were brought back to a floating state. Thereafter, thecells were plated onto two 24-well plates at 37° C. and incubatedovernight. Blasticidin S was added at 1500 μg/ml and then the cells weresubjected to selection culture for 3 to 4 weeks. Total two resistantcolonies obtained by one microcell fusion were isolated, amplified, andsubjected to the following analysis (clone name: DT40 (mChr11-BSr)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] FISH Analysis

DT40 (mChr11-BSr) clones obtained from the above were subjected to FISHanalysis by using mouse Cot-1 DNA as a probe according to the methoddescribed by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175,2001). As a result, 95% of DT40 (mChr11-BSr)-1 had a single copy ofmouse chromosome per normal nuclear type (2n). The following analysiswas performed (FIG. 7).

TABLE 3 Metaphase Interphase mChr11 x0/2n mChr11 x1/2n mChr11 x2/2nmChr11 x2/4n Total x0 x1 x2 x3 Total DT40 1 17 2 20 2 95 3 100(mChr11-BSr)-1 DT40 2 18 20 5 15 79 1 100 (mChr11-BSr)-2[B. 2. 2] Identification of Mouse Chromosome Introduced into DT40 andLabeled with Drug Resistant Gene

SKY-FISH was carried out according to the method described by Kai et al.(Cell Res, 19: 247-58, 2009). As a result, it was found that the mousechromosome introduced into the chicken DT40 cells is mouse chromosome 11(FIG. 8).

[C] Site Specific Cleavage by Telomere Truncation of Distal Region fromMouse Chromosome 11 Region AL671968 in Chicken DT40 Cell

Since less amount of endogenous genes other than a gene to be introducedin the mouse artificial chromosome vector has a less influence on anexperiment system and, among endogenous genes, a gene having aninfluence on development of a mouse individual due to change in geneexpression amount, like an imprinting gene, should be removed as much aspossible, most of the mouse long arm is deleted.

[C. 1] Preparation of Telomere Truncation Vector

As for a basic vector for short arm proximal region-specificrestriction, pBS-TEL/puro construct (Kuroiwa et al. Nature Biotech 2002)was used. From the long-arm proximal nucleotide sequence (AL671968) ofmouse chromosome 11 obtained from GenBank database, target sequence forhomologous recombination was designed. Genomic DNA was extracted fromDT40 (mChr11-BSr) and used as a template, and the sequences of theprimers for PCR amplification of the target sequence for homologousrecombination are given below.

m11 17L: (SEQ ID NO: 3) 5′-CGAGGATCCCACATTGGTAGTCTTTTCACTGCCATCA-3′m11 17R: (SEQ ID NO: 4) 5′-CGAGGATCCCCACTTAACTTTTCCAGGCTTACGGAGA-3′

For PCR, GeneAmp 9600 (manufactured by PerkinElmer, Inc.) was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, and dTTP) used were those included in theproduct and they were used under the conditions described inmanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 65° C. for 8 min were carried out. The PCR product wasdigested with BamHI (TAKARA), separated by agarose gel, purified, andcloned into BamHI site of pBS-TEL/puro (vector name: pBS-TEL/puro_MAC).The targeting vector, target sequence, and chromosome allele obtained byhomologous recombination were shown in FIG. 9.

[C. 2] Selection of Homologous Recombinant

A vector, in which the site specific cleavage of the region distal frommouse chromosome 11 region AL671968 is to be carried out, wastransfected by using pBS-TEL/puro_MAC described above, and puromycinresistant and blasticidin S non-resistant clones were isolated andselected for homologous recombinants. As a result, five clones in whichthe mouse chromosome 11 region could be cleaved were confirmed (clonename: DT40 (MAC)). The targeting vector, target sequence, and chromosomeallele obtained by homologous recombination were shown in FIG. 9.

[C. 3] Selection of Drug Resistant Clone by Mono-Color FISH Analysis

Five DT40 (MAC) clones obtained from the above were subjected to FISHanalysis by using mouse Cot-1 DNA as a probe according to the methoddescribed by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175,2001). As a result, it was confirmed that the long-arm part of mousechromosome 11 was cleaved near the centromere in two clones out of thefive clones (FIG. 10).

TABLE 4 Metaphase Interphase x0/2n x1/2n Total x0 x1 x2 Total Remainingchromosome region Origin DT40 (MAC)-1 2 18 20 94 6 100 No DT40(mChr11-BSr)-1 DT40 (MAC)-2 1 19 20 1 93 6 100 No DT40 (mChr11-BSr)-1DT40 (MAC)-3 1 19 20 1 94 5 100 Yes DT40 (mChr11-BSr)-1 DT40 (MAC)-4 2020 3 93 4 100 Yes DT40 (mChr11-BSr)-1 DT40 (MAC)-5 20 20 100 100 DT40(mChr11-BSr)-1 DT40 (MAC)-6 20 20 5 95 100 No DT40 (mChr11-BSr)-1

From the results above, it was concluded that the mouse artificialchromosome MAC in which extra mouse chromosome long arm has been removedcould be constructed. DT40 (MAC)-1, which is a chicken DT40 cellretaining the mouse artificial chromosome vector MAC, wasinternationally deposited at the National Institute of AdvancedIndustrial Science and Technology (AIST) (Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki, Japan) on May 14, 2009 under the identification nameof DT40 B6bT-1 according to the term of the Budapest Treaty. It wasgiven the accession number FERM BP-11128.

Example 2 Construction of the Mouse Artificial Chromosome Vector MAC1

As a DNA insertion sequence, GFP-PGKneo-loxP-3′ HPRT type loxP sequencewas inserted into the mouse artificial chromosome MAC to construct themouse artificial chromosome vector MAC1. Stability of MAC1 in mouse EScells was examined and stability in each individual tissue was examinedby preparing a progeny-transmitted mouse to which MAC 1 was introduced.

[A] Insertion of GFP-PGKneo-loxP-3′ HPRT Type loxP Sequence into theMouse Artificial Chromosome Vector MAC

[A. 1] Preparation of GFP-PGKneo-loxP-3′ HPRT Type loxP Targeting Vector

As a basic plasmid for inserting loxP sequence into DT40 (MAC), V913(Lexicon genetics) was used. The DNA sequence of mouse chromosome 11 asloxP insertion site was obtained from GenBank database (BX572640.9).From the drug resistant clones, genomic DNA was extracted and used as atemplate, and the sequences of the primers used for amplification of twotarget sequences for homologous recombination are shown below.

m11 5L: (SEQ ID NO: 5) 5′-TGACAGAGAGCTTCCTCCTGCCTCTGTA-3′ m11 5R:(SEQ ID NO: 6) 5′-CTAAAGACCCTCATGCTCCTGTGTGGAA-3′ m11 6L: (SEQ ID NO: 7)5′-GTTCAACCTGAGCTCCACATCATGCTC-3′ m11 7R: (SEQ ID NO: 8)5′-CACTCTTTACCCCTCACCGCTAACCTTG-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 5 min were carried out.

Each of the PCR products was digested with BglII (TAKARA), separated byagarose gel, purified, and cloned into BglII site or BamHI site of V913(vector name: VH21-12). For 3′ HPRT-loxP, loxP sequence obtained byoligosynthesis was cloned into XbaI site of V820 (Lexicon genetics). 3′HPRT-loxP, which is the third to ninth exons of HPRT gene, was clonedbetween EcoRI and AscI of V907 (Lexicon genetics) (vector name: X3.1).Further, the PGKneo sequence cut out by using KpnI and NotI was clonedinto KpnI site and EcoRI site of X3.1 (vector name: X4.1).PGKneo-loxP-3′ HPRT cut out from X4.1 by using KpnI and AscI was clonedinto KpnI site and AscI site of V913 (vector name: pVNLH).HS4-CAG-EGFP-HS4 obtained by digestion with NotI and SalI followed byblunting (provided by Dr. Okabe at Osaka University and Dr. Felsenfeldat NIH) was cloned Into the EcoRV site of pVNLH (vector name: pVGNLH).GFP-PGKneo-loxP-3′ HPRT cassette cut out from pVGNLH with SalI and AscIwas cloned into XhoI site and AscI site of VH21-12 (vector name: pMAC1).The targeting vector, target sequence, and chromosome allele obtained byhomologous recombination are shown in FIG. 11.

[A. 2] Transfection and Isolation of G418 Resistant Clone

Cell culture of chicken DT40 cells was performed in RPMI 1640 culturemedium (Gibco) supplemented with 10% fetal bovine serum (Gibco, hereinbelow, described as FBS), 1% chicken serum (Gibco), and 10-4 M2-mercaptoethanol (Sigma). Approximately 10⁷ DT40 (MAC)-1 cells werewashed once with supplement-free RPMI 1640 culture medium, suspended in0.5 ml of supplement-free RPMI 1640 culture medium, added with 25 μg ofthe targeting vector pMAC1 which has been linearized with therestriction enzyme NotI (TAKARA), transferred to a cuvette (Bio-RadLaboratories, Inc.) for electroporation, and left to stand for 10 min atroom temperature. The cuvette was set in Gene Pulser (Bio-RadLaboratories, Inc.) and voltage was applied under the conditions of 550V and 25 μF. After being left to stand for 10 min at room temperature,the cells were cultured for 24 hours. The culture medium was exchangedwith a culture medium containing G418 (1.5 mg/ml), and dispensed intotwo 96-well culture plates, and then subjected to selection culture forabout 2 weeks. Total 14 resistant colonies obtained after twotransfections were isolated, amplified, and subjected to the followinganalysis (clone name: DT40 (MAC1)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

For extracting genomic DNA from G418 resistant cell line and using it asa template for selecting a recombinant, PCR was carried out by using thefollowing primers and it was confirmed whether or not recombination hassite-specifically occurred on mouse chromosome 11. The primer sequencesare given below.

kj neo: (SEQ ID NO: 9) 5′-CATCGCCTTCTATCGCCTTCTTGACG-3′m11 7R (described above) m11 5L (described above) EGFP-F (L)(SEQ ID NO: 10) 5′-CCTGAAGTTCATCTGCACCA-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,two clones out of the 88 clones were found to be positive for all primersets, and therefore the following analysis was performed by using thosetwo clones.

TABLE 5 Origin DT40 (MAC)-1 Negative Clone name DT40 (MAC1) controlClone number 52 58 DT40 kj neo/m11 7R ∘ ∘ x m11 5L/EGFP-F(L) ∘ ∘ x[A. 3. 2] Two-Color FISH Analysis

For DT40 (MAC1)-52 and DT40 (MAC1)-58 obtained from the above, two-colorFISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out byusing mouse cot-1 DNA and GFP-PGKneo-loxP-3′ HPRT cassette as probes. Asa result, FITC signal derived from the probe was detected near thecentromere of mouse chromosome 11 fragment which has been targeted withloxP sequence, and a signal not observed for the mouse chromosome 11fragment (e.g., DT40 (MAC)-1) before targeting as a negative control wasdetected, therefore it was visually confirmed that recombination hassite-specifically occurred (FIG. 12). From these results, it waspossible to conclude that DT40 cell clones retaining the mouseartificial chromosome vector MAC1 were obtained.

TABLE 6 Metaphase Interphase With FITC x 1/2n Without FITC x 1/2n Totalx0 x1 x2 Total Origin DT40 (MAC)-1 2 18 20 2 95 3 100 Control DT40(MAC1)-52 20 20 98 2 100 DT40 (MAC)-1 DT40 (MAC1)-58 19 1 20 1 95 4 100DT40 (MAC)-1[B] Introduction of MAC1 from DT40 Cell Containing the Mouse ArtificialChromosome Vector MAC1 into CHO Cell

In order to introduce the mouse artificial chromosome vector MAC1 intomouse ES cells via CHO cells, or to stably insert a target gene (group),e.g., CYP3A cluster or the like, via loxP as a DNA sequence insertionsite of the mouse artificial chromosome vector MAC1 within CHO cells,introduction to CHO cells was made.

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (MAC1) 52 and 58 as recipient cells, microcell fusion wascarried out with CHO(HPRT⁻), i.e., CHO hprt depleted cell (obtained fromthe Health Science Research Resources Bank, registration number:JCRB0218), in the same manner as above. Total 24 resistant coloniesobtained by two microcell fusions were isolated, amplified, andsubjected to the following analysis (clone name: CHO(HPRT⁻; MAC1)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from G418 resistant cell line and using it asa template for selecting a recombinant, PCR was carried out by using thefollowing primers and it was confirmed whether or not mouse artificialchromosome MAC1 has been introduced into CHO cells. The primer sequencesare given below.

kj neo (described above)

m11 7R (described above)

m11 5L (described above)

EGFP F (L) (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,20 clones out of the 24 clones were found to be positive for all primersets, and the following analysis was performed by using those 20 clones.

TABLE 7 Origin DT40 (MAC1)-52 DT40 (MAC1)-58 Clone name CHO(HPRT⁻; MAC1)Clone number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 kj neo/m11 7R ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ m11 5L/EGFP-F(L) X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ Origin DT40 (MAC1)-58 Positive control Negative control Clone nameCHO(HPRT⁻; MAC1) DT40(MAC1) Clone number CHO 17 18 19 20 21 22 23 24 5258 DT40 (HPRT) kj neo/m11 7R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X m11 5L/EGFP-F(L) XX X ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X[B. 2. 2] Mono-Color FISH Analysis

20 clones of CHO(HPRT⁻; MAC1) obtained from the above were subjected toFISH analysis by using mouse cot-1 DNA as a probe according to themethod described by Shinohara et al. (Human Molecular Genetics, 10:1163-1175, 2001). As a result, it was confirmed that the mouseartificial chromosome vector MAC1 has been introduced into CHO cellswith a rate of 95% in five clones out of the 20 clones (FIG. 13).

TABLE 8 Metaphase With Origin x0/ x1/ x1/ x2/ x2~/ Big Big Big Bigtranslocation/ Interphase CHO(HPRT⁻; 2n 2n 4n 4n 4n x1/2n x1/4n x2/4nx3~ 2n, 4n Total x0 x1 x2 x3 Total MAC1)- Remarks CHO(HPRT⁻; 1 13 1 1 22 20 3 78 15 4 100 52 MAC1)-2 CHO(HPRT⁻; 1 15 4 20 2 79 16 3 100 52 →hChr7 MAC1)-3 was transferred CHO(HPRT⁻; 8 9 3 20 1 78 18 3 100 52MAC1)-4 CHO(HPRT⁻; 17 3 20 8 83 6 3 100 52 → hChr7 MAC1)-5 wastransferred CHO(HPRT⁻; 1 3 1 10 3 1 1 20 3 67 23 7 100 52 MAC1)-6CHO(HPRT⁻; 2 12 2 1 1 2 20 20 59 14 9 102 52 MAC1)-7 CHO(HPRT⁻; 1 16 320 7 87 6 100 52 → hChr7 MAC1)-8 was transferred CHO(HPRT⁻; 7 2 6 1 4 206 79 7 8 100 52 MAC1)-9 CHO(HPRT⁻; 2 10 3 4 1 20 3 84 9 4 100 52MAC1)-10 CHO(HPRT⁻; 2 18 20 5 39 45 11 100 52 MAC1)-11 CHO(HPRT⁻; 3 1720 9 72 12 7 100 52 MAC1)-12 CHO(HPRT⁻; 1 3 16 20 5 78 9 8 100 58MAC1)-13 CHO(HPRT⁻; 8 4 1 2 5 20 1 83 11 5 100 58 MAC1)-14 CHO(HPRT⁻; 24 6 4 4 20 4 75 13 8 100 58 MAC1)-15 CHO(HPRT⁻; 2 8 4 3 3 20 8 70 21 1100 58 MAC1)-16 CHO(HPRT⁻; 6 1 2 5 5 1 20 2 62 31 5 100 58 MAC1)-20CHO(HPRT⁻; 2 4 2 6 4 2 20 4 72 18 6 100 58 MAC1)-21 CHO(HPRT⁻; 18 1 1 201 91 8 100 58 → hChr7 MAC1)-22 was transferred CHO(HPRT⁻; 13 1 4 2 20 168 29 2 100 58 MAC1)-23 CHO(HPRT⁻; 4 10 3 2 1 20 21 67 10 2 100 58MAC1)-24

From the results above, it was concluded that the mouse artificialchromosome vector MAC1 could be introduced into CHO cells.

[C] Introduction of the Mouse Artificial Chromosome Vector MAC1 from CHOCell Containing the Mouse Artificial Chromosome Vector MAC1 into MouseES Cells

In order to examine the stability of the mouse artificial chromosomevector MAC 1 in mouse ES cells and individual mouse, the mouseartificial chromosome MAC 1 was introduced into mouse ES cells toprepare chimeric mouse and a progeny-transmitted mouse containing themouse artificial chromosome vector MAC1.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

CHO(HPRT⁻; MAC1)-3,5,8, and 22 as recipient cells were cultured on cellculture dishes. At the time of reaching confluency, the culture mediumwas exchanged with F12 culture medium supplemented with 20% FBS and 0.1μg/ml colcemid. After further culturing for 48 hours, the culture mediumwas again exchanged with F12 culture medium supplemented with 20% FBSand 0.1 μg/ml colcemid followed by incubation overnight to formmicrocells. The culture medium was removed and cytochalasin B (10 μg/ml,Sigma) solution which has been previously kept warm at 37° C. was filledin a flask for centrifugation. The centrifugation was performed for 1hour at 34° C., at 8000 rpm. The microcells were suspended in serum freeDMEM culture medium and purified with filters of 8 μm, 5 μm, and 3 μm.After the purification, the cells were centrifuged for 10 min at 2000rpm, and suspended in 5 ml of serum free DMEM culture medium. Themicrocells were suspended in 5 ml of serum free DMEM culture medium andpurified with filters of 8 μm, 5 μm, and 3 μm. After the purification,the cells were centrifuged for 10 min at 2000 rpm.

As a donor cell, B6-ES, which is a C57B6 line-based mouse ES cell, B6(HPRT⁻), which is a HPRT depleted cell line obtained by treating B6-EScell with 6TG, TT2F, which is a C57B6×CBA lineage-based F1 mouse EScell, and KO56 (HPRT⁻), which is a HPRT depleted cell line obtained bytreating TT2F cell with 6TG, were used. For cell culture, to DMEM(Dulbecco's Modified Eagle's Medium-high glucose: SIGMA), 10% FCS, LIF(Muerin Leukemia Inhibitory Factor), 1×10⁻⁵ M 2-ME (2-mercaptoethanol:SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodium pyruvate solution (3.5g/ml: GIBCO), and MEM nonessential amino acids (0.125 mM: GIBCO) wereadded, and culture was performed under 5% CO₂, at 37° C. After washingtwice the cell surface of mouse ES cells with PBS (−), the cells weredispersed with trypsin treatment and recovered with a culture medium inwhich 10% FBS was added to DMEM culture medium. Centrifugation wascarried out at 1500 rpm, the supernatant was removed, re-suspended in 5ml of serum free culture medium, and gently added to the serum freeculture medium containing microcell pellets after centrifugation. It wasfurther centrifuged at 1200 rpm. The supernatant was removed and fusedwith 0.5 ml of PEG1000 (Wako) solution [5 g of PEG1000 is dissolvedcompletely in serum free DMEM culture medium, added with 1 ml ofdimethyl sulfoxide, and sterilized by filtration] precisely for 1 minand 30 sec. 13 ml of serum free culture medium (DMEM) was gently addedand centrifuged at 1200 rpm. The supernatant was removed, common culturemedium for mouse ES cells was added, and using G418 resistant mouseembryonic fibroblast treated with mitomycin as a feeder cell, the cellswere plated onto two culture dishes with a diameter of 10 cm followed byincubation overnight. G418 was added to 250 μg/ml and subjected toselection culture for 3 to 4 weeks (clone name: B6-ES (MAC1) and B6(HPRT⁻; MAC1) and KO56 (HPRT⁻; MAC1)). For B6-ES (MAC1), B6 (HPRT⁻;MAC1) and KO56 (HPRT⁻; MAC1), total 32 resistant colonies obtained bytwo microcell fusions were isolated, amplified, and subjected to thefollowing analysis. For TT2F (MAC1), total 30 resistant coloniesobtained by four microcell fusions were isolated, amplified, andsubjected to the analysis after FISH analysis.

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from G418 resistant cell line and using it asa template for selecting a recombinant, PCR was carried out by using thefollowing primers and it was confirmed whether or not mouse artificialchromosome MAC 1 can be introduced into mouse ES cells. The primersequences are given below.

m11 5L (described above)

EGFP F (L) (described above)

kj neo (described above)

m11 7R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 10 min were carried out. As a result ofPCR, 30 clones out of the 32 clones were found to be positive for allprimer sets, and the following analysis was performed by using 14 clonesamong them.

TABLE 9 Origin CHO(HPRT−; CHO(HPRT−; CHO(HPRT−; CHO(HPRT−; MAC1)-3MAC1)-3 MAC1)-5 CHO(HPRT−; MAC1)-22 MAC1)-3 CHO(HPRT−; MAC1)-22 Clonename B6 (HPRT−; MAC1 B6-ES(MAC1) Clone number 1 2 4 2 3 4 5 6 7 8 9 m115L/ X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ EGFP-F(L) kj neo/m11 7R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯Origin CHO(HPRT−; CHO(HPRT−; CHO(HPRT−; CHO(HPRT−; CHO(HPRT−; CHO(HPRT−;CHO(HPRT−; MAC1)-3 MAC1)-7 MAC1)-22 MAC1)-3 MAC1)-5 MAC1)-7 MAC1)-22Clone name B6-ES(MAC1) KO56(HPRT−; MAC1) Clone number 10 11 12 13 14 1 34 5 6 7 8 9 10 m11 5L/ X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ EGFP-F(L) kj neo/m117R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Origin CHO(HPRT−; CHO(HPRT−; CHO(HPRT−;CHO(HPRT−; CHO(HPRT−; MAC1)-3 MAC1)-7 MAC1)-22 MAC1)-3 MAC1)-22 Clonename KO56(HPRT−; MAC1) Clone number CHO(HPRT−; 11 12 13 14 15 16 17MAC1)-22 CHO(HPRT−) B6-ES TT2F m11 5L/ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X X EGFP-F(L)kj neo/m11 7R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X X[C. 2. 2] Mono-Color FISH Analysis

B6-ES (MAC1) and B6 (HPRT⁻; MAC1) and KO56 (HPRT⁻; MAC1) clones obtainedfrom the above were subjected to FISH analysis by using mouse minorsatellite DNA as a probe according to the method described by Shinoharaet al. (Human Molecular Genetics, 10: 1163-1175, 2001). As a result, itwas confirmed that MAC1 has been introduced into mouse ES cells at arate of 85% or more in all 14 clones. Further, the number of endogenousmouse chromosomes with normal nuclear type was confirmed to be 40 forB6-ES and 39 for KO56. In case of B6 (HPRT⁻; MAC1), clones with 40nuclear type were not obtained. Similarly, for TT2F (MAC1), analysis wascarried out with seven clones. As a result, in all seven clones, it wasconfirmed that the introduction has been made with a rate of 90% ormore. In addition, the number of endogenous mouse chromosomes withnormal nuclear type was confirmed to be 39 for three clones out of theseven clones.

From the results above, it was concluded that the mouse artificialchromosome vector MAC1 can be introduced into mouse ES cells (FIG. 14).

TABLE 10 Number of mouse endogenous chromosomes Origin MAC 38 39 40 41CHO copy number x1 x0 x1 x2 x0 x1 x2 x1 Total MAC1− B6 HPRT−/− 16 1 1 220 22 MAC1-2 B6ES MAC1 3 1 15 4 20 22 B6ES MAC1 8 3 16 1 20 3 B6ES MAC19 1 1 17 1 20 22 KO56 MAC1-1 18 2 20 5 KO56 MAC1-3 1 19 20 7 KO56 MAC1-51 2 16 1 20 22 KO56 MAC1-6 18 2 20 22 KO56 MAC1-7 19 1 20 22 KO56 MAC1-81 19 20 22 KO56 MAC1-9 3 16 1 20 22 KO56 MAC1 10 1 18 1 20 22 KO56 MAC112 1 1 14 2 2 20 7 KO56 MAC1 15 20 20 3

Example 3 Construction of the Mouse Artificial Chromosome VectorCYP3A-MAC

Translocation cloning of CYP3A cluster, which is a group of human drugmetabolizing enzyme-related genes, is carried out for the mouseartificial chromosome vector MAC 1 by using Cre/loxP system to constructCYP3A-MAC. Further, stability of CYP3A-MAC in mouse ES cells is examinedand stability in tissues of each individual is examined by preparing agenetically transmitted progeny mouse into which CYP3A-MAC has beenintroduced. Further, in the genetically transmitted progeny mouse,tissue specific gene expression of CYP3A gene is examined (FIG. 2).

[A] Site Specific Insertion of loxP Sequence into Human Chromosome 7AC004922

For translocation insertion into the mouse artificial chromosome vectorMAC1 via loxP sequence, the loxP sequence is inserted into AC004922proximal to CYP3A gene cluster of human chromosome 7 (hChr7) in DT40cells.

[A. 1] Preparation of Targeting Vector pMPloxPHyg

Targeting vector pMPloxPHyg for inserting loxP, which is a recognitionsequence for Cre recombinase, into AC004922 region, which is locatedextremely close to CYP3A gene locus of human chromosome 7 and on thecentromere side (i.e., locating on the centromere side by approximately300 Kb from CYP3A gene locus), was prepared as follows. First, theAC004922 genome region was amplified by PCR using the following primers.

p450loxP7L; (SEQ ID NO: 11) 5′-GGCCTAGAGCCTGGACTCATTCATTCAA-3′p450loxP7R; (SEQ ID NO: 12) 5′-GACAGATGTCATGCCCCAGGTAGGTATG-3′

As a basic plasmid for inserting loxP sequence, V901 (Lexicon genetics)was used. For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. wasused as a thermal cycler and LA Taq (TAKARA SHUZO CO., LTD.) was used asTaq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used werethose included in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 7 min were carried out.PCR product was treated with proteinase K (Gibco) and subjected to gelfiltration by using CHROMASPIN-TE400 (Clontech). After that, the productwas cleaved with the restriction enzymes BamHI (Boehringer IngelheimGmbH) and EcoRI (NIPPON GENE CO., LTD.) and BglII (NIPPON GENE CO.,LTD.) and subjected to gel filtration by using CHROMASPIN-TE1000(Clontech). The PCR fragments (3.7 kb and 3.0 kb) were cloned into theEcoRI and BamHI or BglII sites of V901 plasmid (vector name: V901-NP21).Next, the V901-NP21 was cleaved with the restriction enzymes AscI (NEB)and KpnI, and from cassette vector 5′ HPRT-loxP-Hyg-TK (Kazuki et al.,Gene Therapy: PMID: 21085194, 2010), the DNA fragment containing loxPwas cut out by using the restriction enzymes AscI and KpnI beforeligation. The resultant product in which the loxP sequence is in thesame direction as the cloned AC004922 genome fragment was taken astargeting vector pMPloxPHyg. Size of the final construct inserted withloxP is 12 kb. The targeting vector, target sequence, and chromosomeallele obtained by homologous recombination are shown (FIG. 15 a).

[A. 2] Transfection and Isolation of Drug Resistant Clone

As described above, targeting vector pMPloxPHyg prepared above waslinearized with the restriction enzyme NotI (TAKARA), and used fortransfection of chicken DT40 cells (clone DF141) retaining humanchromosome 7 fragment (in which the site specific cleavage was made atAF006752 locus) which is prepared by the method described inWO01/011951. After exchanging the culture medium with a culture mediumcontaining hygromycin B (1.5 mg/ml), the cells were dispensed into three96-well culture plates and then subjected to selection culture for about2 weeks. Total 96 resistant colonies obtained from five transfectionswere isolated, amplified, and subjected to the following analysis (clonename: DT40 (hChr7-loxP)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

Genomic DNA was extracted from the hygromycin resistant clone by usingPuregene DNA Isolation Kit (Gentra Systems, Inc.) and identification ofhomologous recombinant was performed by PCR using the following two setsof primer.

Identification of homologous recombinant was performed by PCR using thefollowing two sets of primer.

p450loxP14L; (SEQ ID NO: 13) 5′-AGTTCTTTTGAGGGCCTAGAGCCTGGAC-3′p450loxP14R; (SEQ ID NO: 14) 5′-AAAGGACAGAAGGAGGGAGCAACAGGAT-3′p450loxP16L; (SEQ ID NO: 15) 5′-TCTGGGCATCAGTGTCCTCTCCAGTAAA-3′p450loxP16R; (SEQ ID NO: 16) 5′-TTGGCGACATCCAATGCTAGTGCTATTC-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 4 min were carried out. As a result ofscreening 96 clones, 36 clones were identified as a homologousrecombinant.

[A. 3. 2] Southern Blot Analysis

For the 6 clones which have been confirmed to have recombination by PCRanalysis above, Southern blot analysis was carried out as follows. Thegenomic DNA was treated with the restriction enzyme EcoRI (TAKARA),electrophoresed on 0.8% agarose gel, and subjected to alkali blottingusing a GeneScreen Plus™ hybridization transfer membrane (NENTM LifeScience Products, Inc.). The filter was then subjected to Southernhybridization by using MPp probe, which has been obtained byamplification of the gene sequence in AC004922 by PCR, to identify thehomologous recombinant. For preparing MPp probe, PCR was carried out byusing as a template genomic DNA of DF141 using the primers describedbelow, and ³²P labeled DNA probe was prepared by random priming usingthe PCR product as a template (according to Amersham's attachedprotocols).

Primers for Preparing MPp Probe:

MPp6L; (SEQ ID NO: 17) 5′-TGGAGACGTTGTTTAGCCTCTCCTCCTC-3′ MPp6R;(SEQ ID NO: 18) 5′-CACAGCTTAGAGGCCATTCCCATAGTCC-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and EX Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 93° C. for 5 min, 35 cycles of 93°C. for 1 min, 54° C. for 1 min, and 72° C. for 1 min as one cycle werecarried out. Based on Southern hybridization, it was expected that aband at approximately 10.9 kb is detected from the non-homologousrecombinant while a band at approximately 8.9 kb is detected from thehomologous recombinant (FIG. 15 b). As a result of Southernhybridization, it was found that all 6 clones are the desired homologousrecombinant.

[A. 3. 3] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out forsix clones out of the clones which have been confirmed to haverecombination in the above by using human cot-1 DNA and hygromycin asprobes. As a result, it was confirmed that human chromosome 7 was nottranslocated to the host chromosome in any clone, and based on the factthat hygromycin-derived signal was detected near 7q22, recombination hassite-specifically occurred. From these results, it was concluded thatthe loxP sequence as a gene insertion site was site-specificallyinserted into human chromosome 7 fragment.

[B] Site Specific Cleavage at Human Chromosome 7 Region AC073842 inhChr7-loxP

As disclosed in WO2009/063722 (PCT/JP2008/068928), in order to deletethe genes which are strongly involved with the development of a mouseindividual and exist on the distal side of the CYP3A gene cluster ofhuman chromosome 7, telomere truncation, which is site specific deletionof a chromosome, is performed.

[B. 1] Preparation of Targeting Vector pTELhisD-PT

Targeting vector pTELhisD-PT for inserting human telomere sequence intoAC073842 region, which is located extremely close to CYP3A gene locus ofhuman chromosome 7 and on the telomere side (i.e., locating on thetelomere side by approximately 150 Kb from CYP3A gene locus), wasprepared as follows. First, the AC073842 genome region was amplified byPCR using the following primers.

PT1L; (SEQ ID NO: 19) 5′-TGCGGTGAAGGTCCAAGGAGATAGATTT-3′ PT2R;(SEQ ID NO: 20) 5′-TCTAGCAGAGAGATGGTGGCAGGATTCA-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 20 sec and 68° C. for 8 min were carried out. PCR product wastreated with proteinase K (Gibco) and subjected to gel filtration byusing CHROMASPIN-TE400 (Clontech). After that, the product was cleavedwith the restriction enzymes BamHI (Boehringer Ingelheim GmbH) and BglII(NIPPON GENE CO., LTD.) and subjected to gel filtration by usingCHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the BamHIsite of plasmid pTELhisD (Kuroiwa et al., Nature Biotech., 20: 88,2002). Since the genome sequence direction of AC073842 istelomere→centromere, the resultant product in which cloned genomefragment of AC073842 was in the same direction as the human telomeresequence was taken as desired targeting vector pTELhisD-PT. Size of thefinal construct for long-arm proximal region specific restriction was14.4 kb. The targeting vector, target sequence, and chromosome alleleobtained by homologous recombination are shown in FIG. 16.

[B. 2] Transfection and Isolation of Histidinol Resistant Clone

As described above, targeting vector pTELhisD-PT prepared above waslinearized with the restriction enzyme SrfI (TOYOBO CO., LTD.), and usedfor transfection of clone DT40 (hChr7-loxP) 122 prepared above. Afterexchanging the culture medium with a culture medium containinghistidinol (0.5 mg/ml), the cells were dispensed into ten 96-wellculture plates and then subjected to selection culture for about 2weeks. Total 335 resistant colonies obtained from five transfectionswere isolated, amplified, and subjected to the following analysis (clonename: DT40 (hChr7-loxP-tel)).

[B. 3] Selection of Homologous Recombinant

[B. 3. 1] PCR Analysis

In order to select the recombinant by using genomic DNA of histidinolresistant cell line as a template, as a primary screening, PCR wascarried out by using the following primers that are located closer tothe telomere side than the restriction sites, and it was confirmedwhether or not site specific cleavage has occurred. The primer sequencesare given below.

COPS6-1L; (SEQ ID NO: 21) 5′-TGAGGGTACTTGAAGGGCTGATG-3′ COPS6-1R;(SEQ ID NO: 22) 5′-CAGGGGCTGCTCCCCTTTTATTA-3′ AP4M1-1L: (SEQ ID NO: 23)5′-CCTAACATCGTGTCCCAGCTCA-3′ AP4M1-1R: (SEQ ID NO: 24)5′-TCCTTTCAGACCCCTTCATCTTAG-3′ LRCH4-2L: (SEQ ID NO: 25)5′-TTCAGCCCCAACCAAAGACACTA-3′ LRCH4-1R: (SEQ ID NO: 26)5′-GCCCCGAACCCCTACAAATATAGA-3′ STAG3-1L: (SEQ ID NO: 27)5′-GGGCCTCCAATAAGTGTCCCATA-3′ STAG3-1R: (SEQ ID NO: 28)5′-TTGCTGACTTAGTTGCAGCAGGA-3′ PILRB-2L: (SEQ ID NO: 29)5′-CCCATTGGCAAGATACATGGAGA-3′ PILRB-2R: (SEQ ID NO: 30)5′-AGTGTGGATGCTCCTGGATGAAG-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 95° C. for 10min, 30 cycles of 95° C. for 20 sec, 55° C. for 30 sec, and 72° C. for30 sec were carried out. As a result of PCR, two clones out of the 433clones were found to be positive.

Among the 433 clones, two clones that are not detected with the aboveprimers were confirmed whether site specific homologous recombinationhas occurred by PCR using the following primers. Sequences are asfollows.

PT2R; (described above) hisD2: (SEQ ID NO: 31)5′-GTAAACGCCCTCAAGGAGCAAGCATGA-3′ hisD3: (SEQ ID NO: 32)5′-TGTGACCAAAGATTTAGCGCAGTGCGT-3′

For PCR, LA Taq (TAKARA SHUZO CO., LTD.) was used with the aboveprimers. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 8 min were carried out.Only in two clones having site specific recombination, a band atapproximately 8 kb was detected. In DT40, DT40 (hChr7-loxP) as anegative control, no band was detected.

TABLE 11 Clone name DT40(hChr7-loxP-tel) DT40(hChr7-loxP) Clone number548 608 748 122 DT40 CYP 3A4 R/F ∘ ∘ ∘ ∘ x 3A4 4L/3R ∘ ∘ ∘ ∘ x CYP 3A7R/F ∘ ∘ ∘ ∘ x 3A7 3L/3R ∘ ∘ ∘ ∘ x COPS6 1L/1R ∘ x x ∘ x AP4M 1L/1R ∘ x x∘ x LRCH4 1L/1R ∘ x x ∘ x STAG3 1L/1R ∘ x x ∘ x PILRB 2L/1R ∘ x x ∘ xPILRB 2L/2R ∘ x x ∘ x PT2R/hisD2 x ∘ ∘ x x PT2R/hisD3 x ∘ ∘ x x[B. 3. 2] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out fortwo clones out of the clones which have been confirmed to haverecombination in the above by using human cot-1 DNA and histidinol asprobes. As a result, it was confirmed that human chromosome 7 into whichloxP sequence has been inserted was not translocated to the hostchromosome in any clone, and based on the fact that histidinol-derivedsignal was detected at terminal of human chromosome 7 fragment,recombination has site-specifically occurred.

From these results, it was concluded that, in clone DT40(hChr7-loxP-tel) 608 and 748, cleavage can be made at distal region fromAC073842 which is closer to the telomere side than CYP3A gene clusterregion.

TABLE 12 Metaphase hisD-FITC-stpot x0/2n x1/2n Total + −DT40(hChr7-loxP-tel)-608 3 17 20 17 3 DT40(hChr7-loxP-tel)-748 2 18 2014 6[C] Introduction of hChr7-loxP-tel from DT40 Containing hChr7-loxP-telinto CHO Cell Containing MAC1

For translocation insertion of human CYP3A gene cluster region into themouse artificial chromosome vector MAC1 via loxP sequence in CHO cells,hChr7-loxP-tel is introduced into CHO cells containing the mouseartificial chromosome vector MAC1.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (hChr7-loxP-tel) 608 and 748 as recipient cells, microcellfusion was carried out for CHO(HPRT⁻; MAC1), i.e., a CHO hprt depletedcell containing MAC1 (obtained from the Health Science ResearchResources Bank, registration number: JCRB0218), in the same manner asabove. Total 48 resistant colonies obtained by five microcell fusionswere isolated, amplified, and subjected to the following analysis (clonename: CHO(HPRT⁻; MAC1, hChr7-loxP-tel)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether humanchromosome 7 fragment has been introduced into CHO cells containingMAC1. The primer sequences are given below.

m11 5L (described above) EGFP (F) L (described above)kj neo (described above) m11 6R: (SEQ ID NO: 33)5′-CCCAGGAATCAGTCAGGAAGGCTGTAA-3′ P450 loxP 14L: (described above)hyg F (244): (SEQ ID NO: 34) 5′-GAATTCAGCGAGAGCCTGAC-3′ hyg R (696):(SEQ ID NO: 35) 5′-GATGTTGGCGACCTCGTATT-3′P450 loxP 16R: (described above) CYP3A4 R: (SEQ ID NO: 36)5′-GGCTGCATCAGCATCATCTA-3′ CYP3A4 F: (SEQ ID NO: 37)5′-GCAAGACTGTGAGCCAGTGA-3′ CYP3A5 R: (SEQ ID NO: 38)5′-TCAGCTGTGTGCTGTTGTTTGC-3′ CYP3A5 F: (SEQ ID NO: 39)5′-ATAGAAGGGTCTGTCTGGCTGG-3′ CYP3A7 R: (SEQ ID NO: 40)5′-GAGTTAATGGTGCTAACTGGGG-3′ CYP3A7 F: (SEQ ID NO: 41)5′-ACCCTGAAATGAAGACGGGC-3′ 3A4 4L: (SEQ ID NO: 42)5′-TCCCCCTGAAATTAAGCTTA-3′ 3A4 3R: (SEQ ID NO: 43)5′-TGAGGTCTCTGGTGTTCTCA-3′ 3A7 3L: (SEQ ID NO: 44)5′-TCCCCCTGAAATTACGCTTT-3′ 3A7 3R: (SEQ ID NO: 45)5′-CATTTCAGGGTTCTATTTGT-3′ PT2R: (described above) hisD1:(SEQ ID NO: 46) 5′-GTATTGGTCACCACGGCCGAGTTTCCGC-3′hisD2: (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, DGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,19 clones out of the 48 clones were found to be positive for all primersets, and the following analysis was performed by using 20 clonesincluding 1 negative clone among them.

TABLE 13A Clone origin type A E C D B C Clone name CHO(HPRT⁻; MAC1,hChr7-loxP-tel) Clone number Positive Negative 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 21 35 47 control control m11 5L/ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X EGFP(F)L kj neo/m11 6R ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X P450 loxP 14L/ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X hyg F(244) hyg F(696)/P450 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X loxP 16R CYP3A4 R/F ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X CYP3A5 R/F ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X CYP3A7 R/F ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ X 3A4 4L/3R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X 3A73L/3R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X PT2R/hisD1 ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X X X ◯ X PT2R/hisD2 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X X X ◯ X

TABLE 13B Donor cell line Recipient cell line Origin type ADT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; for each clone MAC1)-5 BDT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; MAC1)-22 CDT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-3 D DT40(hChr7-loxP-tel)-748→ CHO(HPRT⁻; MAC1)-5 E DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-22[C. 2. 2] Two-Color FISH Analysis

19 clones of CHO(HPRT⁻; MAC1, hChr7-loxP-tel) obtained from the abovewere subjected to FISH analysis by using mouse Cot-1 DNA and human Cot-1DNA as probes according to the method described by Shinohara et al.(Human Molecular Genetics, 10: 1163-1175, 2001). As a result, it wasconfirmed that 18 clones except negative clones contain one or twocopies of MAC1 and hChr7-loxP-tel in CHO cells (FIG. 17).

TABLE 14 Metaphase Human copy number hx2 hx1 hx2 hx3 (x1 fragment) hx2Other MAC copy number abnormality mx mx mx mx mx h m including 1/2n 1/2n1/4n 1/2n 2/4n only only translocation Total CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-1 9 2 8 1 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-2 8 1 7 420 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-3 8 5 4 3 20 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-4 8 2 6 4 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-5 12 2 2 11 2 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-6 11 1 8 20 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-7 8 5 2 3 2 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-8 7 2 91 1 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-9 10 4 3 3 20 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-10 8 5 5 2 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-11 13 4 11 1 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-12 5 4 9 2 20 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-13 20 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-14 3 1 6 10 20CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-15 3 17 20 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-16 5 15 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-17 5 15 20CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-18 2 8 10 20 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-40 2 1 17 20 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)47 3 10 4 219 Interphase Human copy number hx1 hx2 hx2 MAC copy number h m mx1 mx1mx2 only only Total Origin CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-1 61 39 100DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-2 75 6 19 100 DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-3 90 4 6 100DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-4 59 32 9 100 DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-5 75 13 12 100DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-6 60 32 8 100 DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-7 63 30 7 100DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-8 39 55 6 100 DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-9 82 17 1 100DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-10 43 53 4 100 DT40(hChr7-loxP-tel)-608 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-11 82 15 3 100DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-22 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-12 33 64 3 100 DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻;MAC1)-3 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-13 57 34 9 100DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-14 42 43 15 100 DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-15 33 47 20 100DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-16 43 36 21 100 DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-17 35 49 16 100DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-5 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-18 32 53 15 100 DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻;MAC1)-5 CHO(HPRT⁻; MAC1, hChr7-loxP-tel)-40 23 77 100DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻; MAC1)-3 CHO(HPRT⁻; MAC1,hChr7-loxP-tel)47 44 42 14 100 DT40(hChr7-loxP-tel)-748 → CHO(HPRT⁻;MAC1)-3

From the results above, it was concluded that hChr7-loxP-tel could beintroduced into CHO cells containing the mouse artificial chromosomevector MAC1.

[D] Site Specific Translocation of 1 Mb Human CYP3A Gene Cluster Region(i.e., AC004922-Human CYP3A Gene Cluster-AC073842) to MAC1 Vector inCHO(HPRT⁻; MAC1, hChr7-loxP-tel) Clone

To stably keep the human CYP3A gene cluster, which is a DNA with 1 Mbsize, in an mouse individual, translocation insertion into the mouseartificial chromosome vector MAC1 was performed (FIG. 18).

[D. 1] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection for CHO(HPRT⁻; MAC1,hChr7-loxP-tel)-6,9,12, and 47 obtained from the above. To cells in 6wells with 90% confluency, 3 μg of Cre was introduced according to thecommercially available protocol (Invitrogen). After culture for 2 weeksunder HAT selection culture, a resistant colony was generated and total42 colonies obtained by four introductions were isolated, amplified, andsubjected to the following analysis (clone name: CHO (CYP3A-MAC1,hChr7-ΔCYP3A)).

[D. 2] Selection of Drug Resistant Clone

[D. 2. 1] PCR Analysis

For extracting genomic DNA from HAT resistant cell line and using it asa template for selecting a clone with reciprocal translocation, PCR wascarried out by using the following primers and it wad confirmed whetheror not reciprocal chromosomal translocation has occurred on humanchromosome 7 fragment and MAC1. The primer sequences are given below.

P450 loxP 16R (described above) hyg R (696) (described above)kj neo (described above) P450 loxP 14L (described above)m11 5L (described above) m11 6R (described above)CYP3A4 R (described above) CYP3A4 F (described above)CYP3A5 R (described above) CYP3A5 F (described above)CYP3A7 R (described above) CYP3A7 F (described above)3A4 4L (described above) 3A4 3R (described above)3A7 3L (described above) 3A7 3R (described above) TRANS L1:(SEQ ID NO: 47) 5′-TGGAGGCCATAAACAAGAAGAC-3′ TRANS R1: (SEQ ID NO: 48)5′-CCCCTTGACCCAGAAATTCCA-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,27 clones out of the 42 clones were found to be positive for all primersets, and therefore the following analysis was performed by using those27 clones.

TABLE 15 Cell origin 9 6 12 47 12 Clone name CHO(CYP3A-MAC1 +hChr7-ΔCYP3A) Clone number 3 10 11 12 13 14 15 16 18 19 20 22 23 24 2526 27 28 P450 loxP16R/hygR(696) ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ kjneo/P450 loxP 14L ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ m11 5L/hyg R(696)◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ P450 loxP16R/TRANS-R1 ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Trans L1/m11 6R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ Trans L1/R1 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ CYP3A4 R/F ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ CYP3A5 R/F ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯CYP3A7 R/F ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ 3A4 4L/3R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ 3A7 3L/3R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Cellorigin 12 47 12 12 Control Clone name CHO(CYP3A-MAC1 + hChr7-ΔCYP3A) CHOClone number (HPRT⁻; MAC1, CHO 31 32 33 34 35 37 40 41 42hChr7-loxP-tel)12 (HPRT⁻) P450 loxP16R/hygR(696) ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Xkj neo/P450 loxP 14L ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X m11 5L/hyg R(696) ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ X NT P450 loxP16R/TRANS-R1 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X NT Trans L1/m11 6R◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X Trans L1/R1 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X CYP3A4 R/F ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ X CYP3A5 R/F ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X CYP3A7 R/F ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ X 3A4 4L/3R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X 3A7 3L/3R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯X[D. 2. 2] Two-Color FISH Analysis

27 clones of CHO (CYP3A-MAC1, hChr7-ΔCYP3A) obtained from the above weresubjected to FISH analysis by using mouse Cot-1 DNA and human Cot-1 DNAas probes according to the method described by Shinohara et al. (HumanMolecular Genetics, 10: 1163-1175, 2001). As a result, it was confirmedthat 25 clones out of the 27 clones are observed at a rate of 50% ormore with the signal derived from human chromosome 7 on MAC1 whichconsists of mouse chromosome 11 fragment containing loxP sequence (FIG.19).

TABLE 16 With MAC Total number of human chromosome One Two Three ThreeSix Human region-retaining MAC MAC(+Rho MAC(+Rho MAC(+Rho MAC(+Rho MAC(weak MAC(+Rho spot) x1 spot) x2 spot) x1 spot) x1 or no Rho) x1 spot)x2 MAC region-retaining hChr7f +h7(+FITC +h7(+FITC +h7(+FITC +h7(+FITC+h7(+FITC +h7(+FITC spot) spot) spot) x1 spot) x1 spot) x1 spot)x2 x1 x2+h7(noFITC) +h7(noFITC) +h7(no FITC) +h7(no FITC) /2n /4n x1/2n x2/2nx2/2n x4/4n CHO(CYP3A-MAC + hChr7-Δ CYP3A)-3 CHO (CYP3A-MAC + hChr7-ΔCYP3A)-10 CHO(CYP3A-MAC + hChr7-Δ 14 CYP3A)-11 CHO(CYP3A-MAC + hChr7-Δ13 CYP3A)-12 CHO(CYP3A-MAC + hChr7-Δ 17 CYP3A)-13 CHO(CYP3A-MAC +hChr7-Δ 12 CYP3A)-14 CHO(CYP3A-MAC + hChr7-Δ 16 CYP3A)-15CHO(CYP3A-MAC + hChr7-Δ 17 3 CYP3A)-16 CHO(CYP3A-MAC + hChr7-Δ 14 3CYP3A)-18 CHO(CYP3A-MAC + hChr7-Δ 17 3 CYP3A)-19 CHO(CYP3A-MAC + hChr7-Δ11 5 4 CYP3A)-20 CHO(CYP3A-MAC + hChr7-Δ 20 CYP3A)-22 CHO(CYP3A-MAC +hChr7-Δ 16 2 CYP3A)-23 CHO(CYP3A-MAC + hChr7-Δ 4 14 1 CYP3A)-24CHO(CYP3A-MAC + hChr7-Δ 7 8 2 CYP3A)-25 CHO(CYP3A-MAC + hChr7-Δ 16 2 1CYP3A)-26 CHO(CYP3A-MAC + hChr7-Δ 10 9 1 CYP3A)-27 CHO(CYP3A-MAC +hChr7-Δ 12 3 1 CYP3A)-28 CHO(CYP3A-MAC + hChr7-Δ 3 12 5 CYP3A)-31CHO(CYP3A-MAC + hChr7-Δ 11 5 CYP3A)-32 CHO(CYP3A-MAC + hChr7-Δ 13 1CYP3A)-33 CHO (CYP3A-MAC + hChr7-Δ 17 CYP3A)-34 CHO(CYP3A-MAC + hChr7-Δ12 1 3 1 CYP3A)-35 CHO(CYP3A-MAC + hChr7-Δ 16 1 CYP3A)-37CHO(CYP3A-MAC + hChr7-Δ 14 CYP3A)-40 CHO(CYP3A-MAC + hChr7-Δ 10 2 1CYP3A)-41 CHO(CYP3A-MAC + hChr7-Δ 13 CYP3A)-42 Without MAC Total numberof human chromosome One Two Three Human region-retaining MAC h7(+FITCh7(+FITC spot)x1+ spot)x1+ Origin MAC region-retaining hChr7f CHO h7 h7(HPRT⁻; h7 (+FITC) (wihtout (without MAC1, spot) x 1 FITC FITC AbnormalNo hChr7-I only spot)x1 spot)x2 translocation translocation TotaloxP-tel) CHO(CYP3A-MAC + hChr7-Δ 19 1 20 9 CYP3A)-3 CHO (CYP3A-MAC +hChr7-Δ 19 1 20 6 CYP3A)-10 CHO(CYP3A-MAC + hChr7-Δ 6 20 12 CYP3A)-11CHO(CYP3A-MAC + hChr7-Δ 7 20 12 CYP3A)-12 CHO(CYP3A-MAC + hChr7-Δ 3 2047 CYP3A)-13 CHO(CYP3A-MAC + hChr7-Δ 8 20 47 CYP3A)-14 CHO(CYP3A-MAC +hChr7-Δ 4 20 47 CYP3A)-15 CHO(CYP3A-MAC + hChr7-Δ 20 47 CYP3A)-16CHO(CYP3A-MAC + hChr7-Δ 3 20 47 CYP3A)-18 CHO(CYP3A-MAC + hChr7-Δ 20 47CYP3A)-19 CHO(CYP3A-MAC + hChr7-Δ 20 47 CYP3A)-20 CHO(CYP3A-MAC +hChr7-Δ 20 12 CYP3A)-22 CHO(CYP3A-MAC + hChr7-Δ 2 20 12 CYP3A)-23CHO(CYP3A-MAC + hChr7-Δ 1 20 12 CYP3A)-24 CHO(CYP3A-MAC + hChr7-Δ 3 2012 CYP3A)-25 CHO(CYP3A-MAC + hChr7-Δ 1 20 12 CYP3A)-26 CHO(CYP3A-MAC +hChr7-Δ 20 12 CYP3A)-27 CHO(CYP3A-MAC + hChr7-Δ 4 20 12 CYP3A)-28CHO(CYP3A-MAC + hChr7-Δ 20 12 CYP3A)-31 CHO(CYP3A-MAC + hChr7-Δ 4 20 12CYP3A)-32 CHO(CYP3A-MAC + hChr7-Δ 6 20 12 CYP3A)-33 CHO (CYP3A-MAC +hChr7-Δ 3 20 47 CYP3A)-34 CHO(CYP3A-MAC + hChr7-Δ 3 20 47 CYP3A)-35CHO(CYP3A-MAC + hChr7-Δ 3 20 12 CYP3A)-37 CHO(CYP3A-MAC + hChr7-Δ 6 2012 CYP3A)-40 CHO(CYP3A-MAC + hChr7-Δ 7 20 12 CYP3A)-41 CHO(CYP3A-MAC +hChr7-Δ 7 20 12 CYP3A)-42

From these results, it was concluded that 1 Mb of CYP3A cluster on humanchromosome 7 fragment into which loxP sequence has been inserted couldbe cloned into the mouse artificial chromosome vector MAC1 by reciprocaltranslocation.

Example 4 Construction of the Mouse Artificial Chromosome Vector MAC2

The mouse artificial chromosome vector MAC2 is constructed in which 5′HPRT-loxP-PGKhyg type loxP sequence, which is a DNA insertion sequence,is inserted into the mouse artificial chromosome vector MAC (FIG. 3). Asthe 5′ HPRT-loxP-PGK hyg type loxP sequence is inserted to HAC vectorcarrying GFP (21HAC2) derived from chromosome 21 described in a reportby Kazuki et al. (Gene Therapy: PMID: 21085194, 2010), gene expressionof HAC and MAC can be compared to each other in the same vector.Further, the gene introduction vector for insertion into 21HAC2 can beused as it is without undergoing the step of preparing a vector.

[A] Insertion of 5′ HPRT-loxP-PGKhyg Type loxP Sequence into MouseArtificial Chromosome MAC

[A. 1] Preparation of 5′ HPRT-loxP-PGKhyg Type loxP Targeting Vector

As a basic plasmid for inserting loxP sequence, VH21-12 prepared abovewas used. The 5′ HPRT-loxP-PGKhygro cassette cut out from the X6.1 byusing KpnI and AscI was cloned into KpnI and AscI sites of V907 (Lexicongenetics) (vector name: p V907-AML). Further, 5′ HPRT-loxP-PGK hygrocassette was cut out from p V907-AML by using XhoI and SalI and clonedinto XhoI site of VH21-12 (vector name: pMAC2). The targeting vector,target sequence, and chromosome allele obtained by homologousrecombination are shown in FIG. 20.

[A. 2] Transfection and Isolation of Drug Resistant Clone

As described above, targeting vector pMAC2 prepared above was linearizedwith the restriction enzyme NotI (TAKARA), and used for transfection ofclone DT40 (MAC) prepared above. After exchanging the culture mediumwith a culture medium containing hygromycin (1.5 mg/ml), the cells weredispensed into two 96-well culture plates and then subjected toselection culture for about 2 weeks. Total 45 resistant coloniesobtained from one transfection were isolated, amplified, and subjectedto the following analysis (clone name: DT40 (MAC2)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cells and using itas a template for selecting a recombinant, PCR was carried out by usingthe following primers and it was confirmed whether or not recombinationhas site-specifically occurred on the mouse artificial chromosome vectorMAC. The primer sequences are given below.

TRANS-L (described above) m11 6R (described above)m11 7R (described above) m11 4L: (SEQ ID NO: 49)5′-ACTCCTAAGGGAGTTGGTGCTGTTGGTG-3′ m11 5L (described above)hygF (244): (described above) hygR (696): (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,eight clones out of the 45 clones were found to be positive for allprimer sets. Six clones randomly selected from those eight clones weresubjected to the following analysis.

TABLE 17 TRANS-L1/ TRANS-L1/ hygF(244)/ hygF(244)/ m114L/ m115L/ m116Rm117R m116R m117R hygR(696) hygR(696) Cell origin DT40(MAC2)-1 X NT NTNT NT NT DT40 (MAC)-1 DT40(MAC2)-2 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-3 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-4 ◯ ◯ ◯ ◯ ◯ ◯ DT40(MAC)-1 DT40(MAC2)-5 ◯ ◯ ◯ ◯ ◯ ◯ DT40 (MAC)-1 DT40(MAC2)-6 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-7 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-8X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-9 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-10 ◯ ◯ ◯ ◯ ◯ ◯ DT40 (MAC)-1 DT40(MAC2)-11 X NT NT NT NT NTDT40 (MAC)-1 DT40(MAC2)-12 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-13 ◯◯ ◯ ◯ ◯ ◯ DT40 (MAC)-1 DT40(MAC2)-14 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-15 ◯ ◯ ◯ ◯ ◯ ◯ DT40 (MAC)-1 DT40(MAC2)-16 X NT NT NT NT NTDT40 (MAC)-1 DT40(MAC2)-17 ◯ ◯ ◯ ◯ ◯ ◯ DT40 (MAC)-1 DT40(MAC2)-18 X NTNT NT NT NT DT40 (MAC)-1 DT40(MAC2)-19 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-20 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-21 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-22 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-23 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-24 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-25 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-26 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-27 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-28 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-29 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-30 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-31 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-32 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-33 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-34 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-35 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-36 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-37 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-38 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-39 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-40 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-41 X NT NT NT NT NT DT40 (MAC)-1 DT40(MAC2)-42 X NT NT NT NTNT DT40 (MAC)-1 DT40(MAC2)-43 X NT NT NT NT NT DT40 (MAC)-1DT40(MAC2)-44 ◯ ◯ ◯ ◯ ◯ ◯ DT40 (MAC)-1 DT40(MAC2)-45 ◯ ◯ ◯ ◯ ◯ ◯ DT40(MAC)-1 Positive control ◯ NT NT NT NT NT Negative control X X X X X X[A. 3. 2] Two-Color FISH Analysis

For the 6 clones of DT40 (MAC2) obtained from the above, two-color FISHanalysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out byusing mouse cot-1 DNA and 5′ HPRT-loxP-PGK hygro cassette as probes. Asa result, it was found that the detection rate of a signal clearlyderived from probe is 10% in the mouse artificial chromosome vector MAC,which is a mouse chromosome 11 fragment before targeting as negativecontrol, while it is detected with a rate of 50% or more a signalderived from probe in the six clones of DT40 (MAC2). Thus, site specificrecombination occurring in the six clones was visually confirmed (FIG.21). From these results, it was possible to conclude that DT40 cellclones retaining the mouse artificial chromosome vector MAC2 areobtained.

TABLE 18 Metaphase Interphase MACx1 F−/2n MACx1 F+/2n MACx1 F weak+/2nMACx2/4n Total x0 x1 x2 Total DT40(MAC)-1 12 2 5 1 20 100 100DT40(MAC2)-2 1 15 3 1 20 98 2 100 DT40(MAC2)-5 18 2 20 97 3 100DT40(MAC2)-17 1 18 1 20 2 96 2 100 DT40(MAC2)-10 4 16 20 3 94 3 100DT40(MAC2)-13 2 14 4 20 2 98 100 DT40(MAC2)-15 6 10 4 20 5 92 3 100[B] Introduction of MAC2 from Chicken DT40 Cell Containing the MouseArtificial Chromosome Vector MAC2 into CHO Cells

In order to stably introduce the mouse artificial chromosome vector MAC2into mouse ES cells, introduction to CHO cells is carried out. Further,in order to stably insert a target gene (for example, GFP gene) vialoxP, which is a DNA insertion site of the mouse artificial chromosomevector MAC2, introduction to CHO cells is carried out.

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (MAC2)-5 and 17 as a recipient cell, microcell fusion wascarried out for CHO(HPRT⁻), which is CHO hprt depleted cells (obtainedfrom the Health Science Research Resources Bank, registration number:JCRB0218), in the same manner as above. Total 44 resistant coloniesobtained by two microcell fusions were isolated, amplified, andsubjected to the following analysis (clone name: CHO(HPRT⁻; MAC2)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether or not mouseartificial chromosome MAC2 can be introduced into CHO cells. The primersequences are given below.

TRANS-L (described above)

m11 6R (described above)

m11 7R (described above)

m11 4L (described above)

m11 5L (described above)

hygF (244): (described above)

hygR (696): (described above))

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,14 clones out of the 44 clones were found to be positive for all primersets, and the following analysis was performed by using clones randomlyselected from those 14 clones.

TABLE 19 Cell origin DT40(MAC2)-17 DT40(MAC2)-5 DT40(MAC2)-17 Clone nameCHO(HPRT; MAC2) Clone number 11 12 13 14 15 16 17 18 19 20 21 22 23 24Positive control Negative control TRANS-L1/m11 6R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ X TRANS-L1/m11 7R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X hyg F(244)/m116R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X hyg F(244)/m11 7R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ X m11 4L/hygR(696) ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X m115L/hygR(696) ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X[B. 2. 2] Two-Color FISH Analysis

With nine clones randomly selected from the 14 clones of CHO(HPRT⁻;MAC2) obtained from the above, FISH analysis was carried out by usingmouse Cot-1 DNA and 5′ HPRT-loxP-PGK hygro cassette as probes by themethod according to Matsubara et al. (FISH test protocol, Shujunsha Co.,Ltd., 1994). As a result, it was confirmed that MAC2 was introduced intoCHO cells with a rate of 95% in eight clones out of the nine clones(FIG. 22).

TABLE 20 Metaphase CHO nuclear type 2n MAC copy number Interphase 0 1 23 4 1 2 3 Total x0 x1 x2 x3 x4 x5 Total Origin DT40 (MAC)-6 20 20CHO(HPRT⁻; MAC2)-12 19 19 2 88 10 100 DT40(MAC2)-5 CHO(HPRT⁻; MAC2)-1320 20 0 91 9 100 DT40(MAC2)-5 CHO(HPRT⁻; MAC2)-14 1 12 4 2 1 20 12 46 357 100 DT40(MAC2)-5 CHO(HPRT⁻; MAC2)-15 1 17 1 1 20 3 79 16 2 100DT40(MAC2)-5 CHO(HPRT⁻; MAC2)-16 1 2 4 12 1 20 1 15 55 23 4 2 100DT40(MAC2)-5 CHO(HPRT⁻; MAC2)-17 13 4 1 1 19 1 70 27 2 100 DT40(MAC2)-17CHO(HPRT⁻; MAC2)-18 1 17 2 20 14 72 13 1 100 DT40(MAC2)-17 CHO(HPRT⁻;MAC2)-22 1 13 5 1 20 7 69 17 6 1 100 DT40(MAC2)-17 CHO(HPRT⁻; MAC2)-23 415 1 20 26 64 9 1 100 DT40(MAC2)-17

From these results, it was concluded that the mouse artificialchromosome vector MAC2 in which loxP sequence as a gene insertion siteis inserted into mouse artificial chromosome MAC, which is a chromosomefragment derived from mouse chromosome 11, could be introduced into CHOcells.

[C] Introduction of the Mouse Artificial Chromosome Vector MAC2 from CHOCell Containing the Mouse Artificial Chromosome Vector MAC2 into MouseES Cell.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

CHO(HPRT⁻; MAC2)-13 and -18 as recipient cells were cultured on cellculture dishes. At the time of reaching confluency, the culture mediumwas exchanged with F12 culture medium supplemented with 20% FBS and 0.1μg/ml colcemid. After further culturing for 48 hours, the culture mediumwas again exchanged with F12 culture medium supplemented with 20% FBSand 0.1 μg/ml colcemid followed by incubation overnight to formmicrocells. The culture medium was removed, and cytochalasin B (10μg/ml, Sigma) solution which has been previously kept warm at 37° C. wasfilled in a flask for centrifugation. The centrifugation was performedfor 1 hour at 34° C., at 8000 rpm. The microcells were suspended inserum free DMEM culture medium and purified with filters of 8 μm, 5 μm,and 3 μm. After the purification, the cells were centrifuged for 10 minat 2000 rpm, and suspended in 5 ml of serum free DMEM culture medium.The microcells were suspended in 5 ml of serum free DMEM culture mediumand purified with filters of 8 μm, 5 μm, and 3 μm. After thepurification, the cells were centrifuged for 10 min at 2000 rpm.

As a donor cell, B6-ES, which is a C57B6 line-based mouse ES cellobtained from CLEA Japan, Inc., B6 (HPRT⁻), which is a HPRT depletedcell line obtained by treating the ES cell with 6TG, and KO56 (HPRT⁻),which is a HPRT depleted cell line of TT2F cell, were used. For cellculture, to DMEM (Dulbecco's Modified Eagle's Medium-high glucose:SIGMA), 10% FCS, LIF (Muerin Leukemia Inhibitory Factor), 1×10⁻⁵ M 2-ME(2-mercapto-ethanol: SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodiumpyruvate solution (3.5 g/ml: GIBCO), and MEM nonessential amino acid(0.125 mM: GIBCO) were added and culture was performed at 5% CO₂, 37° C.After washing twice the cell surface of mouse ES cells with PBS (−), thecells were dispersed with trypsin treatment and recovered with culturemedium in which 10% FBS was added to DMEM culture medium. Centrifugationwas carried out at 1500 rpm, the supernatant was removed andre-suspended in 5 ml of serum free culture medium and gently added tothe serum free culture medium containing pellets of microcells aftercentrifugation. It was further centrifuged at 1200 rpm. The supernatantwas removed and fused with 0.5 ml of PEG1000 (Wako) solution [5 g ofPEG1000 is dissolved completely in serum free DMEM culture medium, 1 mlof dimethyl sulfoxide is added thereto, and the mixture is sterilized byfiltration] precisely for 1 min and 30 sec. 13 ml of serum free culturemedium (DMEM) was gently added and centrifuged at 1200 rpm. Thesupernatant was removed, common culture medium for mouse ES cells wasadded, and by using G418 resistant mouse embryonic fibroblast treatedwith mitomycin as a feeder cell, the cells were plated onto two cellculture dishes with a diameter of 10 cm followed by incubationovernight. Hygromycin was added so as to be 250 μg/ml and selectionculture was carried out for 3 to 4 weeks. Total 28 resistant coloniesobtained by two microcell fusions were isolated, amplified, andsubjected to the following analysis (clone name: B6-ES (MAC2), B6(HPRT⁻; MAC2), and KO56 (HPRT⁻; MAC2)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether or not mouseartificial chromosome MAC2 can be introduced into mouse ES cells. Theprimer sequences are given below.

TRANS L (described above)

m11 6R (described above)

m11 4L (described above)

hyg R (696) (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 10 min were carried out. As a result ofPCR, 27 clones out of the 28 clones were found to be positive for allprimer sets, and the following analysis was performed by using threeclones randomly selected from those 27 clones.

TABLE 21 Origin CHO(HPRT−; MAC2)-13 CHO(HPRT−; KO56 Clone nameKO56(HPRT−; MAC2) MAC2)-13 (HPRT−) Clone number 1 2 3 + − TRANS-L1/ ∘ ∘∘ ∘ x m11 6R hyg F(244)/ ∘ ∘ ∘ ∘ x m11 6R m11 4L/hygR ∘ ∘ ∘ ∘ x (696)[C. 2. 2] Mono-Color FISH Analysis

With the mouse ES (MAC2) clones obtained from the above, FISH analysiswas carried out according to the method described by Shinohara et al.(Human Molecular Genetics, 10: 1163-1175, 2001) by using mouse minorsatellite DNA as a probe. As a result, it was confirmed that, in oneclone out of the three clones, one copy of MAC2 is introduced into mouseES cells at a rate of 80% or more and the number of endogenous mousechromosomes that are normal nuclear type of KO56 cells was 39.

From these results, it was concluded that the mouse artificialchromosome vector MAC2 in which loxP sequence, which is a gene insertionsite, was inserted into mouse artificial chromosome MAC, which is achromosome fragment derived from mouse chromosome 11, could beintroduced into mouse ES cells (FIG. 23).

TABLE 22 Mouse chromosome 36 37 38 39 40 +MAC copy number x0 x1 x1 x1 x0x1 Total KO56(HPRT−; MAC2)-2 1 1 3 14 1 20[D] As described in Example 8, in vitro stability can be examined byusing mouse ES cell retaining the mouse artificial chromosome vectorMAC2. Further, by preparing a chimeric mouse using the ES cells, themouse lineage-based TC (MAC2) in which MAC2 is genetically transmittedto a progeny can be prepared. Still further, by using the TC (MAC2)mouse line, stability of MAC2 in somatic cells can be examined.

Example 5 Construction of the Mouse Artificial Chromosome Vector MAC3

The mouse artificial chromosome vector MAC3 is constructed in which thePGKneo-loxP-3′ HPRT type loxP sequence as a DNA insertion sequence isinserted into mouse artificial chromosome MAC (FIG. 4). Stability of themouse artificial chromosome vector MAC3 in mouse ES cells is examined,and by preparing a genetically transmitted progeny mouse to which MAC3has been introduced, stability in individual tissue is examined.

[A] Insertion of PGKneo-loxP-3′ HPRT Type loxP Sequence into MouseArtificial Chromosome MAC

[A. 1] Preparation of PGKneo-loxP-3′ HPRT Type loxP Targeting Vector

As a basic plasmid for inserting loxP sequence, VH21-12 prepared abovewas used. The PGKneo-loxP-3′ HPRT cassette cut out from pVNLH by usingSalI and AscI was cloned into XhoI and AscI sites of VH21-12 (vectorname: pMAC3). The targeting vector, target sequence, and chromosomeallele obtained by homologous recombination are shown in FIG. 24.

[A. 2] Transfection and Isolation of G418 Resistant Clone

Cell culture of chicken DT40 cells was performed in RPMI 1640 culturemedium (Gibco) supplemented with 10% fetal bovine serum (Gibco, hereinbelow, described as FBS), 1% chicken serum (Gibco), and 10⁻⁴ M2-mercaptoethanol (Sigma). Approximately 10⁷ DT40 (MAC)-1 cells werewashed once with supplement-free RPMI 1640 culture medium, suspended in0.5 ml of supplement-free RPMI 1640 culture medium, added with 25 μg ofthe targeting vector pMAC3 which has been linearized with therestriction enzyme NotI (TAKARA), transferred to a cuvette (Bio-RadLaboratories, Inc.) for electroporation, and left to stand for 10 min atroom temperature. The cuvette was set in Gene Pulser (Bio-RadLaboratories, Inc.) and applied with voltage under the conditions of 550V and 25 μF. After left to stand for 10 min at room temperature, thecells were cultured for 24 hours. The culture medium was exchanged witha culture medium containing G418 (1.5 mg/ml), and dispensed into two96-well culture plates, and then subjected to selection culture forabout 2 weeks. Total 14 resistant colonies obtained after twotransfections were isolated, amplified and subjected to the followinganalysis (clone name: DT40 (MAC3)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

For extracting genomic DNA from G418 resistant cell line and using it asa template for selecting a recombinant, PCR was carried out by using thefollowing primers and it was confirmed whether or not recombination hassite-specifically occurred on mouse chromosome 11. The primer sequencesare given below.

m11 17L (described above) Puro-1: (SEQ ID NO: 50)5′-GAGCTGCAAGAACTCTTCCTCACG-3′ kj neo (described above)m11 6R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they are used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 9 min were carried out. As a result of PCR,16 clones out of the 17 clones were found to be positive for all primersets, and therefore, the following analysis was performed by using twoclones randomly selected from those 16 clones.

TABLE 23 DT40 (MAC3) Telomere DT40 33 49 56 75 100 109 111 130 157 160184 187 197 308 330 331 407 vector MAC1 DT40 m11 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ NT X 17L/ Puro-1 kj neo/ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X ◯ ◯ ◯NT ◯ X m11 6R[A. 3. 2] Mono-Color FISH Analysis

With the two clones of DT40 (MAC3) obtained from the above, FISHanalysis was carried out by using mouse Cot-1 DNA as a probe accordingto the method described by Shinohara et al. (Human Molecular Genetics,10: 1163-1175, 2001). As a result, it was found that no chromosometranslocation or the like occurred in any clone and it was retainedindependently with a rate of 90% or more.

TABLE 24 Metaphase Interphase MACx1/2n Total x0 x1 x2 Total RemarksDT40(MAC3)- 20 20 4 96 100 →MMCT 160 into CHO DT40(MAC3)- 20 20 2 98 100→MMCT 187 into CHO[A. 3. 3] Two-Color FISH Analysis

With the randomly selected DT40 (MAC3)-160 and 187, two-color FISHanalysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out byusing mouse cot-1 DNA and mouse minor satellite DNA as probes. As aresult, it was found that mouse artificial chromosome MAC3 wasindependently present in single copy (FIG. 25).

From these results, it was concluded that DT40 cell clones retaining themouse artificial chromosome vector MAC3 in which loxP sequence as a DNAinsertion sequence was inserted near the mouse centromere were obtained.

[B] Introduction of MAC3 from Chicken DT40 Cell Containing the MouseArtificial Chromosome Vector MAC3 into CHO Cell

In order to stably insert a target gene (for example, GFP gene) via loxPas a DNA sequence insertion site of the mouse artificial chromosomevector MAC3, introduction to CHO cells was carried out.

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

DT40 (MAC3)-160 as a recipient cell was cultured on a cell culture dish.At the time of reaching confluency, the culture medium was exchangedwith RPMI 1640 culture medium supplemented with 20% FBS, 1% chickenserum, 10-4 M 2-mercaptoethanol, and 0.05 μg/ml colcemid and culturedfurther for 12 hours to form microcells. The culture medium was replacedwith 24 ml of serum free DMEM culture medium, and 2 ml of them wasdispensed into 12 25 cm² flasks for centrifugation which has been coatedin advance with 100 μm1 poly L-lysine and cultured for 30 min at 37° C.to adhere the cells at the bottom of the flask. The supernatant wasremoved and cytochalasin B (10 μg/ml, Sigma) solution which has beenpreviously kept warm at 37° C. was filled in a flask for centrifugationfollowed by centrifugation for 1 hour at 34° C., 8000 rpm. Themicrocells were suspended in serum free DMEM culture medium and purifiedwith filters of 8 μm, 5 μm, and 3 μm. After the purification, cells werecentrifuged for 10 min at 1700 rpm and then suspended in 5 ml of serumfree DMEM culture medium.

As a donor cell, CHO(HPRT⁻), which is CHO hprt depleted cells (obtainedfrom the Health Science Research Resources Bank, registration number:JCRB0218) was used. Purified micronuclei were re-suspended in 2 ml ofserum free culture medium containing PHA-P (SIGMA), and gently platedonto CHO cells from which culture supernatant [F12 culture mediumsupplemented with 10% FBS (Invitrogen)] was removed. The plate wasincubated for 15 min at 37° C. The supernatant was removed and fusedwith 1 ml of PEG1000 (Wako) solution [5 g of PEG1000 is completelydissolved in serum free DMEM culture medium, added with 1 ml of dimethylsulfoxide, and sterilized by filtration] precisely for 1 min. The cellswere washed four times with 4 ml of serum free culture medium (DMEM),added with 5 ml of common culture medium for CHO cells, and incubatedovernight. Cell surfaces were washed twice with PBS (−) and the cellswere dispersed by trypsin treatment, and plated onto five cell culturedishes with a diameter of 10 cm. After adding G418 so as to be 800μg/ml, cells were subjected to selection culture for 3 to 4 weeks. Total12 resistant colonies obtained by two microcell fusions were isolated,amplified, and subjected to the following analysis (clone name:CHO(HPRT⁻; MAC3)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from G418 resistant cell line and using it asa template for selecting a recombinant, PCR was carried out by using thefollowing primers and it was confirmed whether or not the mouseartificial chromosome vector MAC3 can be introduced into CHO cells. Theprimer sequences are given below.

kj neo (described above)

m11 6R (described above)

m11 17L (described above)

Puro-1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 10 min were carried out. As a result ofPCR, six clones out of the seven clones were found to be positive forall primer sets, and the following analysis was performed by using thosesix clones.

TABLE 25 Derived from DT40 (MAC3)-160 Positive control Negative controlNegative control CHO(HPRT−; MAC3) DT40 DT40 CHO 1 3 5 6 7 9 11(MAC3)-160 (MAC)-1 HPRT−/− kj-neo/m11 6R ◯ X ◯ ◯ ◯ ◯ ◯ ◯ X X m1117L/Puro-1 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X X[B. 2. 2] Mono-Color FISH Analysis

With the six clones of CHO(HPRT⁻; MAC3) obtained from the above, FISHanalysis was carried out by using mouse Cot-1 DNA as a probe accordingto the method described by Shinohara et al. (Human Molecular Genetics,10: 1163-1175, 2001). As a result, it was confirmed that MAC3 wasintroduced into CHO cells with a rate of 90% or more in three clones outof the six clones.

TABLE 26 Metaphase With Interphase x0/2n x1/2n x1/4n x2/2n x2/4n x2/8nx3/4n translocation Total x0 x1 x2 x3 Total Origin CHO(HPRT−; MAC3)-1 412 2 1 1 20 25 64 9 2 100 DT40(MAC3)-160 CHO(HPRT−; MAC3)-5 2 17 1 20 2760 10 3 100 DT40(MAC3)-160 CHO(HPRT−; MAC3)-6 6 8 1 1 1 1 2 20 29 56 114 100 DT40(MAC3)-160 CHO(HPRT−; MAC3)-7 2 16 1 1 20 16 66 13 5 100DT40(MAC3)-160 CHO(HPRT−; MAC3)-9 2 8 1 5 1 3 20 12 58 20 10 100DT40(MAC3)-160 CHO(HPRT−; MAC3)-11 5 5 2 3 5 20 12 54 26 8 100DT40(MAC3)-160

From these results, it was concluded that the mouse artificialchromosome vector MAC3 could be introduced into CHO cells.

[C] Introduction of MAC3 from CHO Cell Containing the Mouse ArtificialChromosome Vector MAC3 into Mouse ES Cell

To examine stability of the mouse artificial chromosome vector MAC3 inmouse ES cells and a mouse individual, mouse artificial chromosome MAC3is introduced into mouse ES cells, and the chimeric mouse andgenetically transmitted progeny mouse containing mouse artificialchromosome vector MAC3 are prepared.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

CHO(HPRT⁻; MAC3)-1 and -6 as recipient cells were cultured on cellculture dishes. At the time of reaching confluency, the culture mediumwas exchanged with F12 culture medium supplemented with 20% FBS and 0.1μg/ml colcemid. After further culturing for 48 hours, the medium culturewas exchanged with F12 culture medium supplemented with 20% FBS and 0.1μg/ml colcemid followed by incubation overnight to form microcells. Theculture medium was removed and cytochalasin B (10 μg/ml, Sigma) solutionwhich has been previously kept warm at 37° C. was filled in a flask forcentrifugation. The centrifugation was performed for 1 hour at 34° C.,at 8000 rpm. The microcells were suspended in serum free DMEM culturemedium and purified with filters of 8 μm, 5 μm, and 3 μm. After thepurification, the microcells were centrifuged for 10 min at 2000 rpm,and suspended in 5 ml of serum free DMEM culture medium. The microcellswere suspended in 5 ml of serum free DMEM culture medium and purifiedwith filters of 8 μm, 5 μm, and 3 μm. After the purification, the cellswere centrifuged for 10 min at 2000 rpm.

As a donor cell, B6 (HPRT⁻), which is HPRT depleted cell line obtainedby treating ES cells derived from a mouse C57B6 lineage obtained fromCLEA Japan, Inc. with 6TG, was used. For cell culture, to DMEM(Dulbecco's Modified Eagle's Medium-high glucose: SIGMA), 10% FCS, LIF(Muerin Leukemia Inhibitory Factor), 1×10⁻⁵ M 2-ME (2-mercaptoethanol:SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodium pyruvate solution (3.5g/ml: GIBCO), and MEM nonessential amino acid (0.125 mM: GIBCO) wereadded and culture was performed at 5% CO₂, 37° C. After washing twicethe cell surface of mouse ES cells with PBS (−), the cells weredispersed with trypsin treatment and recovered with culture medium inwhich 10% FBS was added to DMEM culture medium. Centrifugation wascarried out at 1500 rpm, the supernatant was removed and re-suspended in5 ml of serum free culture medium and gently added to the serum freeculture medium containing pellets of microcells after centrifugation. Itwas further centrifuged at 1200 rpm. The supernatant was removed andfused with 0.5 ml of PEG1000 (Wako) solution [5 g of PEG1000 isdissolved completely in serum free DMEM culture medium, added with 1 mlof dimethyl sulfoxide, and sterilized by filtration] precisely for 1 minand 30 sec. 13 ml of serum free culture medium (DMEM) was gently addedand centrifuged at 1200 rpm. The supernatant was removed, common culturemedium for mouse ES cells was added, and by using G418 resistant mouseembryonic fibroblast treated with mitomycin as a feeder cell, the cellswere plated onto two cell culture dishes with a diameter of 10 cmfollowed by incubation overnight. G418 was added so as to be 250 μg/mland selection culture was carried out for 3 to 4 weeks. Total 28resistant colonies obtained by two microcell fusions were isolated,amplified, and subjected to the following analysis (clone name: B6(HPRT⁻; MAC3)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from the G418 resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers, and it was confirmed whether or not sitespecific cleavage has occurred on mouse chromosome 11. The primersequences are given below.

kj neo (described above)

m11 6R (described above)

m11 17L (described above)

Puro-1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 10 min were carried out. As a result ofPCR, 27 clones out of the 28 clones were found to be positive for allprimer sets, and the following analysis was performed by using those 27clones.

TABLE 27 Origin CHO(HPRT−; MAC3)-6 CHO(HPRT−; MAC3)5 CHO(HPRT⁻; MAC3)-1Clone name B6 (HPRT⁻; MAC3) B6(HPRT⁻; MAC3) Clone number 1 2 3 4 5 6 7 8s1 s2 s3 s4 s5 s6 s7 s8 kj neo/m11 6R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯m11 17L/Puro-1 ◯ ◯ ◯ ◯ ◯ X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Origin CHO(HPRT⁻; MAC3)-1Clone name B6(HPRT⁻; MAC3) Positive Negative Clone number controlcontrol s9 s10 s11 s12 s13 s14 s15 s16 s17 s18 s19 s20 CHO H−/− B6-ES kjneo/m11 6R ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ (MAC3)-5 HPRT−/− m11 17L/Puro-1 ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X[C. 2. 2] Mono-Color FISH Analysis

For the 16 clones of B6 (HPRT⁻; MAC3) obtained from the above, FISHanalysis was carried out by using mouse minor satellite DNA as a probeaccording to the method described by Shinohara et al. (Human MolecularGenetics, 10: 1163-1175, 2001). As a result, it was confirmed that MAC3was introduced into the mouse ES cells with a rate of 95% or more infive clones out of the 16 clones.

From these results, it was concluded that the mouse artificialchromosome vector MAC3 could be introduced into mouse ES cells (FIG.27).

TABLE 28 Metaphase 38 + 39 + 39 + 40 + 40 + 40 + MAC 40 + 41 + 41 + 41 +MACx1/ MACx1 0 MACx1 0 MACx1 fragmentx1 MACx2 MACx1 MACx2 0 4n TotalOrigin B6 3 1 1 2 7 CHO(HPRT⁻; MAC3)-6 (HPRT⁻; MAC3)-1 B6 2 16 2 20CHO(HPRT⁻; MAC3)-6 (HPRT⁻; MAC3)-2 B6 1 1 1 16 1 20 CHO(HPRT⁻; MAC3)-6(HPRT⁻; MAC3)-3 B6 3 17 20 CHO(HPRT⁻; MAC3)-5 (HPRT⁻; MAC3)-4 B6 6 2 4 44 20 CHO(HPRT⁻; MAC3)-5 (HPRT⁻; MAC3)-5 B6 1 16 2 1 20 CHO(HPRT⁻;MAC3)-5 (HPRT⁻; MAC3)-7 B6 1 2 16 1 20 CHO(HPRT⁻; MAC3)--1 (HPRT⁻;MAC3)-s1 B6 8 12 20 CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)-s2 B6 20 20CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)-s3 B6 1 19 20 CHO(HPRT⁻; MAC3)--1(HPRT⁻; MAC3)-s4 B6 2 6 10 2 20 CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)-s5 B61 1 17 1 20 CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)-s6 B6 4 8 8 20 CHO(HPRT⁻;MAC3)--1 (HPRT⁻; MAC3)-s7 B6 20 20 CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)-s8B6 1 1 8 2 8 20 CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)-s9 B6 1 2 17 20CHO(HPRT⁻; MAC3)--1 (HPRT⁻; MAC3)- s10[D] Stability of the Mouse Artificial Chromosome Vector MAC3 in Mouse ESCells

Under non-selection culture of 0 to 100 PDL for the mouse ES clonesobtained from the above (for example, B6 (HPRT⁻; MAC3)-3, -s6, obtainedfrom the above [C]), the rate of cells retaining MAC1 after long termculture was measured by FISH analysis. As a result, the retention rateof 95% or more was obtained even for 100 PDL (FIG. 28).

From these results, it was confirmed that the mouse artificialchromosome vector MAC3 is very stably maintained at a rate of 95% ormore in mouse ES cells (in vitro).

[E] Preparation of Chimeric Mouse Retaining the Artificial ChromosomeVector MAC3

By using the ES cell clones obtained from the above, a chimeric mousewas prepared according to the technique in (Gene Targeting, ExperimentalMedicine, 1995). As a host cell, the morula obtained by sexualcrossbreeding of MCH (ICR) (white, purchased from CLEA Japan, Inc.) wasused. Injected embryo is transplanted into a foster mother, and coatcolor of the new-born mouse is examined to see whether or not it is achimera. Further, the chimeric rate can be determined from theintracellular contribution of ES cells for forming an individual in ICRembryonic cells.

As a result of transplanting 60 embryos injected with B6 (HPRT; MAC3)clone (for example, B6 (HPRT; MAC3)-ES (MAC3)-s6 obtained from theabove) into a foster mother, eight chimeric mice (dark brown color areais observed in coat color) were born. Among eight animals, two weremales in which one was 10% chimeric mouse and the other one was 5%chimeric mouse and six were females in which four were 10% chimericmouse and the remaining two were 5% chimeric mouse. In other words, itwas shown that ES cell line (B6 HPRT−/− cell line) retaining mouseartificial chromosome MAC3 retains a chimera forming ability, that is,an ability of differentiating into normal tissues of a mouse individual.

[F] As described in Example 8, mouse line-based TC (MAC3) in which MAC3is transmitted to a progeny can be prepared by crossbreeding a chimericmouse retaining the mouse artificial chromosome vector MAC3 and a wildtype mouse. Further, by using TC (MAC3) mouse line, stability of MAC3 insomatic cells can be examined.

Example 6 Construction of the Mouse Artificial Chromosome Vector GFP-MAC

As an example of a gene encoding useful proteins, EGFP as a fluorescencegene is inserted into the mouse artificial chromosome vector MAC3 byusing Cre/loxP system and expression of functional protein and long-termstability are examined (FIG. 5).

[A] Insertion of Specific Gene (for example, GFP) into the MouseArtificial Chromosome Vector MAC3 by Using Cre/loxP System in CHO CellContaining Mouse Artificial Chromosome Vector MAC3

It is examined whether or not loxP is operated and plasmid DNA can besite-specifically inserted for the mouse artificial chromosome vectorMAC3 obtained by inserting PGKneo-loxP-3′ HPRT type loxP sequence as aDNA insertion sequence into mouse artificial chromosome MAC.

[A. 1] Preparation of EGFP Insertion Vector

As a basic plasmid for inserting loxP sequence, V913 (Lexicon genetics)was used. In 5′ HPRT-loxP, loxP sequence obtained by oligo synthesis wascloned into XbaI site of V820 (Lexicon genetics). 5′ HPRT-loxP wascloned into ClaI and AscI of V907 (Lexicon genetics), and PGKhygro wascloned into ClaI and KpnI sites (vector name: pX6.1). Further, into theNotI site and SalI site of X6.1, HS4-CAG-EGFP-HS4 cut out by using NotIand SalI (provided by Dr. Okabe at Osaka University and Dr. Felsenfeldat NIH) was cloned to give a GFP-inserted construct for HPRTreconstruction system (vector name: pX6.1 EGFP). Chromosome sitespecific DNA insertion obtained by GFP insertion in HPRT reconstructionsystem based on Cre/loxP system is given in FIG. 29.

[A. 2] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection. To cells in 6 wellswith 90% confluency, 1 μg of Cre and 2 μg of GFP insertion vector wereintroduced according to the commercially available protocol(Invitrogen). After culture for 2 weeks under HAT selection culture, aresistant colony was generated and total 22 colonies obtained by twointroductions were isolated, amplified, and subjected to the followinganalysis (clone name: CHO (GFP-MAC)).

[A. 3] Selection of Drug Resistant Clone

[A. 3. 1] Confirmation of GFP Insert According to FluorescenceMicroscope Observation

22 cloned colonies were observed under a fluorescence microscope, and asa result, all clones were observed to have GFP positive cells, and thepositive rate was almost 100%.

[A. 3. 2] PCR Analysis

In order to select a recombinant by using genomic DNA of HAT resistantcell line as a template, PCR was carried out by using the followingprimers, and it was confirmed whether or not site specific insertion ofGFP gene has occurred. The primer sequences are given below.

TRANS L1 (described above)

TRANS R1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 10min, 35 cycles of 94° C. for 30 sec, 60° C. for 30 sec, and 72° C. for30 sec were carried out. As a result of PCR, all 22 clones were found tobe positive and, the following analysis was performed by using those 22clones.

TABLE 29 Origin Negative CHO(HPRT⁻; MAC3)-5 CHO(HPRT⁻; MAC3)-6 controlClone name CHO(GFP-MAC) Clone number CHO 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 20 21 22 (HPRT) TRANS L1/R1 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ X[A. 3. 3] Two-Color FISH Analysis

For the randomly selected six clones, two-color FISH analysis wascarried out according to Matsubara et al. (FISH test protocol, ShujunshaCo., Ltd., 1994). FISH analysis was carried out by using mouse cot-1 DNAand X6.1EGFP as probes. As a result, it was found that, in three clonesout of the six clones, a single copy of GFP-MAC was retained at a rateof 50% or more and signal derived from X6.1EGFP was generated. Since nosignal was detected from MAC3 before site specific insertion of EGFP asa negative control, it was confirmed that EGFP was site-specificallyinserted (FIG. 30).

TABLE 30 Metaphase X1 X1 x2 x2 x2 x2 x3 x3 F(−)/ F(+)/ F(+, +) F(+, −)F(−, −) F(+, −)/ (+, +, +)/ (+, +, +)/ x0/2n 2n 2n 4n 4n 4n 2n 2n 4nCHO(HPRT⁻; MAC)-5 5 13 1 1 CHO(GFP-MAC)-2 2 7 9 2 CHO(GFP-MAC)-4 2 1 10CHO(GFP-MAC)-5 2 5 1 CHO(GFP-MAC)-8 1 CHO(GFP-MAC)-10 3 13 1 1 1CHO(GFP-MAC)-12 3 13 2 1 Metaphase With Interphase translocation Totalx0 x1 x2 x3 Total Origin CHO(HPRT⁻; MAC)-5 20 22 63 13 2 100CHO(GFP-MAC)-2 20 22 71 2 5 100 CHO(HPRT⁻; MAC3)-5 CHO(GFP-MAC)-4 7 20 779 10 4 100 CHO(HPRT⁻; MAC3)-5 CHO(GFP-MAC)-5 12 20 9 81 5 5 100CHO(HPRT⁻; MAC3)-5 CHO(GFP-MAC)-8 19 20 4 32 42 22 100 CHO(HPRT⁻;MAC3)-5 CHO(GFP-MAC)-10 1 20 32 63 3 2 100 CHO(HPRT⁻; MAC3)-5CHO(GFP-MAC)-12 1 20 8 76 14 2 100 CHO(HPRT⁻; MAC3)-5

From the above experiments, it was confirmed that GFP expression wasobserved based on the fact that the mouse artificial chromosome MAC3carried the GFP gene, and as a result, CHO cells retaining the mouseartificial chromosome vector GFP-MAC were obtained.

[B] Introduction of GFP-MAC from CHO Cell Containing the MouseArtificial Chromosome Vector GFP-MAC to Mouse ES Cell

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

CHO (GFP-MAC)-4, -10, and -12 as recipient cells were cultured on cellculture dishes. At the time of reaching confluency, the culture mediumwas exchanged with F12 culture medium supplemented with 20% FBS and 0.05μg/ml colcemid. After further culturing for 48 hours, the culture mediumwas exchanged with F12 culture medium supplemented with 20% FBS and 0.05μg/ml colcemid followed by incubation overnight to form microcells. Theculture medium was removed and cytochalasin B (10 μg/ml, Sigma) solutionwhich has been previously kept warm at 37° C. was filled in a flask forcentrifugation. The centrifugation was performed for 1 hour at 34° C.,8000 rpm. The microcells were suspended in serum free DMEM culturemedium and purified with filters of 8 μm, 5 μm, and 3 μm. After thepurification, the cells were centrifuged for 10 min at 2000 rpm, andsuspended in 5 ml of serum free DMEM culture medium.

The microcells were suspended in 5 ml of serum free DMEM culture mediumand purified with filters of 8 μm, 5 μm, and 3 μm. After thepurification, the cells were centrifuged for 10 min at 2000 rpm.

As a donor cell, wild type B6 cells, which were established from EScells derived from a mouse C57B6 lineage obtained from CLEA Japan, Inc.and mouse ES cells of wild type TT2F cell, were used. For cell culture,DMEM (Dulbecco's Modified Eagle's Medium-high glucose: SIGMA)supplemented with 10% FCS, LIF (Muerin Leukemia Inhibitory Factor),1×10⁻⁵ M 2-ME (2-mercaptoethanol: SIGMA), L-glutamine (3.5 g/ml: GIBCO),sodium pyruvate solution (3.5 g/ml: GIBCO), and MEM nonessential aminoacid (0.125 mM: GIBCO) and culture was performed in the presence of 5%CO₂ at 37° C. After washing twice the cell surface of mouse ES cellswith PBS (−), the cells were dispersed with trypsin treatment, andrecovered with culture medium in which 10% FBS was added to DMEM culturemedium. Centrifugation was carried out at 1500 rpm, the supernatant wasremoved, re-suspended in 5 ml of serum free culture medium, and gentlyadded to the serum free culture medium containing pellets of microcellsafter centrifugation. It was further centrifuged at 1200 rpm. Thesupernatant was removed and fused with 0.5 ml of PEG1000 (Wako) solution[5 g of PEG1000 is dissolved completely in serum free DMEM culturemedium, added with 1 ml of dimethyl sulfoxide, and sterilized byfiltration] precisely for 1 min and 30 sec. 13 ml of serum free culturemedium (DMEM) was gently added and centrifuged at 1200 rpm. Thesupernatant was removed, common culture medium for mouse ES cells wasadded, and by using G418 resistant mouse embryonic fibroblast treatedwith mitomycin as a feeder cell, the cells were plated onto two cellculture dishes with a diameter of 10 cm followed by incubationovernight. G418 was added to 250 μg/ml and selection culture was carriedout for 3 to 4 weeks. Total 36 resistant colonies obtained by twomicrocell fusions were isolated, amplified, and subjected to thefollowing analysis (clone name: TT2F (GFP-MAC) and B6-ES (GFP-MAC)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from G418 resistant cell line and using it asa template for selecting a recombinant, PCR was carried out by using thefollowing primers, and it was confirmed whether or not site specificcleavage has occurred on mouse chromosome 11. The primer sequences aregiven below.

TRANS L1: (described above)

TRANS R1: (described above)

m11 6R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions for TRANSL1/R1 were as follows: after heat denaturation at 94° C. for 1 min, 35cycles of 98° C. for 10 sec and 68° C. for 1 min were carried out.Temperature and cycle conditions for TRANS L1/ml 1 6R were as follows:after heat denaturation at 94° C. for 1 min, 35 cycles of 98° C. for 10sec and 68° C. for 7 min were carried out. As a result of PCR, 34 clonesout of the 36 clones were found to be positive for all primer sets, andthe following analysis was performed by using 24 clones randomlyselected from the positive clones.

TABLE 31 CHO (GFP-MAC)- TRANS TRANS Clone name • Number derived cloneL1/R1 L1/m11 6R TT2F(GFP-MAC)-1 4 ∘ ∘ TT2F(GFP-MAC) -2 4 ∘ ∘TT2F(GFP-MAC) -3 4 ∘ ∘ TT2F (GFP-MAC) -4 4 ∘ ∘ TT2F (GFP-MAC) -5 4 ∘ ∘TT2F (GFP-MAC) -6 4 ∘ ∘ TT2F (GFP-MAC) -7 4 ∘ ∘ TT2F (GFP-MAC) -8 4 ∘ ∘TT2F (GFP-MAC) -9 4 ∘ ∘ TT2F (GFP-MAC) -10 10 ∘ ∘ TT2F (GFP-MAC) -11 4 ∘∘ TT2F (GFP-MAC) -12 4 ∘ ∘ TT2F (GFP-MAC) -13 4 ∘ ∘ TT2F (GFP-MAC) -14 4∘ ∘ TT2F (GFP-MAC) -15 10 Δ ∘ TT2F (GFP-MAC) -16 10 ∘ ∘ TT2F (GFP-MAC)-17 10 ∘ ∘ TT2F (GFP-MAC) -18 10 ∘ ∘ TT2F (GFP-MAC) -19 10 ∘ ∘ TT2F(GFP-MAC) -20 10 ∘ ∘ TT2F (GFP-MAC) -21 10 ∘ ∘ TT2F (GFP-MAC) -22 12 ∘ ∘B6-ES (GFP-MAC) -1 4 ∘ ∘ B6-ES (GFP-MAC) -2 4 ∘ ∘ B6-ES (GFP-MAC) -3 4 ∘∘ B6-ES (GFP-MAC) -4 4 ∘ ∘ B6-ES (GFP-MAC) -5 4 ∘ ∘ B6-ES (GFP-MAC) -6 4∘ ∘ B6-ES (GFP-MAC) -7 10 ∘ x B6-ES (GFP-MAC) -8 10 ∘ ∘ B6-ES (GFP-MAC)-9 10 ∘ ∘ B6-ES (GFP-MAC) -10 10 ∘ ∘ B6-ES (GFP-MAC)-11 10 ∘ ∘ B6-ES(GFP-MAC) -12 4 ∘ ∘ B6-ES (GFP-MAC) -14 12 ∘ x CHO (GFP-MAC)-10 Positive∘ ∘ control TT2F Negative x x control B6-ES Negative x x control[B. 2. 2] Quinacrine-Hoechst Double Staining

Clones found to be positive by the above PCR analysis were subjected toQuinacrine-Hoechst double staining by the same method described above.Chromosome images of the clone obtained after Quinacrine-Hoechst doublestaining were fluorescence-microscopically observed, and as a result itwas found that 18 clones out of the 24 clones retained the mouseartificial chromosome GFP-MAC at a rate of 100%.

TABLE 32 Without MAC Metaphase With MAC Metaphase 39 + 40 + 35 + 38 +38 + 39 + 39 + 39 + 40 + 40 + 41 + 42 + 43 + 0 0 Mx1 Mx1 Mx2 Mx1 Mx2 Mx3Mx1 Mx2 Mx1 Mx1 Mx1 Total TT2F(GFP-MAC)-1 1 1 7 1 10 TT2F(GFP-MAC)-2 2 16 1 10 TT2F(GFP-MAC)-3 2 1 1 6 10 TT2F(GFP-MAC)-5 1 9 10 TT2F(GFP-MAC)-64 6 10 TT2F(GFP-MAC)-7 2 3 5 10 TT2F(GFP-MAC)-10 4 6 10 TT2F(GFP-MAC)-117 3 10 TT2F(GFP-MAC)-12 10 10 TT2F(GFP-MAC)-13 1 2 5 1 1 10TT2F(GFP-MAC)-15 3 7 10 TT2F(GFP-MAC)-16 2 4 2 2 10 TT2F(GFP-MAC)-17 1 36 10 B6-ES(GFP-MAC)-1 5 5 10 B6-ES(GFP-MAC)-2 1 9 10 B6-ES(GFP-MAC)-3 18 1 10 B6-ES(GFP-MAC)-4 3 7 10 B6-ES(GFP-MAC)-5 1 9 10 B6-ES(GFP-MAC)-61 7 2 10 B6-ES(GFP-MAC)-8 10 10 B6-ES(GFP-MAC)-9 10 10 B6-ES(GFP-MAC)-102 3 5 10 B6-ES(GFP-MAC)-11 10 10 B6-ES(GFP-MAC)-12 6 4 10

From these results, it was concluded that the mouse ES cells to whichmouse artificial chromosome GFP-MAC has been introduced had a normalnuclear type and could be used for long-term culture and preparation ofa chimeric mouse.

[B. 2. 3] Two-Color FISH Analysis

With mouse ES (GFP-MAC) clones obtained from the above, FISH analysiswas carried out by using mouse minor satellite DNA and pX6.1E as probesaccording to the method described by Shinohara et al. (Human MolecularGenetics, 10: 1163-1175, 2001). As a result, it was confirmed thatGFP-MAC has been introduced into mouse ES cells at a rate of 95% or morein six clones out of the 12 clones.

TABLE 33 Metaphase Number of mouse chromosomes 35 36 37 38 39 40 MACcopy number Mx4 Mx1 Mx0 Mx1 Mx2 Mx4 Mx0 Mx1 Mx2 Mx4 Mx0 Mx1 Mx2 Mx0B6ES(GFP-MAC)-4 1 5 B6ES (GFP-MAC)-5 3 2 B6ES(GFP-MAC)-8 1 1 2B6ES(GFP-MAC)-9 2 2 B6ES(GFP-MAC)-11 1 1 16 B6ES(GFP-MAC)-12 1 3TT2F(GFP-MAC)-1 1 2 2 1 12 1 TT2F(GFP-MAC)-2 2 2 2 13 TT2F(GFP-MAC)-3 35 1 9 TT2F(GFP-MAC)-11 2 1 7 9 TT2F(GFP-MAC)-12 1 1 2 16TT2F(GFP-MAC)-13 2 1 1 2 8 3 Metaphase Number of mouse chromosomes 40 4144 45 78 MAC copy number Mx1 Mx2 Mx3 Mx4 Mx4 Mx4 Mx1 Mx2 Total OriginB6ES(GFP-MAC)-4 13 1 20 CHO(GFP-MAC)-4 B6ES (GFP-MAC)-5 15 20CHO(GFP-MAC)-4 B6ES(GFP-MAC)-8 14 1 1 20 CHO(GFP-MAC)-10 B6ES(GFP-MAC)-916 20 CHO(GFP-MAC)-10 B6ES(GFP-MAC)-11 2 20 CHO(GFP-MAC)-10B6ES(GFP-MAC)-12 6 8 2 20 CHO(GFP-MAC)-4 TT2F(GFP-MAC)-1 1 20CHO(GFP-MAC)-4 TT2F(GFP-MAC)-2 1 20 CHO(GFP-MAC)-4 TT2F(GFP-MAC)-3 1 120 CHO(GFP-MAC)-4 TT2F(GFP-MAC)-11 1 20 CHO(GFP-MAC)-4 TT2F(GFP-MAC)-1220 CHO(GFP-MAC)-4 TT2F(GFP-MAC)-13 1 18 CHO(GFP-MAC)-4

From these results, it was concluded that the mouse artificialchromosome vector GFP-MAC could be introduced into mouse ES cells.

[C] Stability of the Mouse Artificial Chromosome Vector GFP-MAC in MouseES Cells

Under non-selection culture of 0 to 100 PDL for the mouse ES clonesobtained from the above (for example, B6-ES (MAC3)-9, obtained from theabove [B]), the rate of cells retaining GFP-MAC3 after long-term culturewas measured by FISH analysis. As a result, the retention rate of 95% ormore was obtained even for 100 PDL (FIG. 31 and FIG. 32). The colonieswere also observed under fluorescence microscope and all clones wereobserved to have GFP positive cells, and the positive rate was almost100%.

TABLE 34 Number of mouse chromosomes 38 39 40 41 42 MAC copy number x0x1 x0 x1 x2 x0 x1 x0 x1 x1 Total MAC retention rate B6ES(GFP-MAC)-9 0PDL1 1 18 20 95% B6ES(GFP-MAC)-9 50PDL With drug selection 1 17 2 20 90%Without drug selection 1 14 1 4 20 95% B6ES(GFP-MAC)-9 100PDL With drugselection 3 7 10 100% Without drug selection 1 3 12 3 1 20 100%

From these results, it was concluded that an exogenous gene having 20 kbor less (for example, EGFP gene) could be site-specifically andefficiently inserted into the mouse artificial chromosome vector MAC3 byusing Cre/loxP system, and the MAC3 carrying the exogenous gene was verystable in mouse ES cells and the expression of the exogenous gene onMAC3 was stable for a long period of time.

[D] Preparation of Chimeric Mouse Retaining Artificial Chromosome VectorGFP-MAC

By using the ES cell clones obtained from the above [B], a chimericmouse was prepared according to the known techniques (Gene Targeting,Experimental Medicine, 1995). As a host cell, the morula and eight-cellstage embryo obtained by sexual crossbreeding of MCH (ICR) (white,purchased from CLEA Japan, Inc.) were used. Injected embryo wastransplanted into a foster mother, and coat color of the new-born mousecould be examined to see whether or not it is a chimera.

As the result that embryos (260 wild type male B6 (GFP-MAC) clones and180 wild type female TT2F (GFP-MAC) clones) injected with wild type maleB6 (GFP-MAC) clone and wild type (GFP-MAC) TT2F female clone (forexample, B6-ES (GFP-MAC) 4 and 18, TT2F (GFP-MAC)-12, which are obtainedfrom the above) were transplanted into foster mothers, chimeric mice(dark brown color area was observed in coat color) were born. 42chimeric mice derived from male wild type B6 (GFP-MAC) clone were born,and 20 of them were male mice, in which one was GFP-positive 50%chimeric mouse, five were 40% chimeric mouse, one was 30% chimericmouse, seven were 20% chimeric mouse, three were 10% chimeric mouse, andthree were 5% chimeric mouse. Further, 14 chimeric mice derived fromwild type TT2F (GFP-MAC) clone were born, and one of them was aGFP-positive individual having a chimeric rate of about 100% from whichalmost no white color area was observed.

From the above results, it was shown that ES cell line (B6 and TT2F)retaining the mouse artificial chromosome vector GFP-MAC had a chimeraforming ability, that is, an ability of differentiating into normaltissues of a mouse individual.

[E] Transmission to Progeny of Mouse Artificial Chromosome from ChimericMouse Retaining the Mouse Artificial Chromosome Vector GFP-MAC

Among four new-born mice born from a chimeric mouse obtained bycrossbreeding between the female chimeric mouse (chimeric rate of about100%) prepared from the above [D] and the C57B6 (black, purchased fromCLEA Japan, Inc.) male mouse, three were observed with fluorescence ofGFP, which is a dominant genetic trait of GFP-MAC derived from the EScells. Further, one new-born mouse among the three animals was observedwith fluorescence of GFP from the entire body, and therefore it wasfound that the mouse artificial chromosome is stable in individual mouse(FIG. 33). The mouse line in which GFP-MAC has been transmitted to aprogeny is referred to as TC (GFP-MAC). As described in Example 8,stability of GFP-MAC in somatic cells could be examined by using the TC(GFP-MAC) mouse line.

Example 7 Stability of the Mouse Artificial Chromosome Vector MAC1

[A. 1] Stability of the Mouse Artificial Chromosome Vector MAC1 in CHOCell

Under non-selection culture of 0 to 25 PDL for the CHO clones (forexample, CHO (HPRT⁻; MAC1)-8 and -22, obtained from Example 2 above)obtained from the above, the rate of cells retaining MAC1 afterlong-term culture was measured by FISH analysis. As a result, theretention rate of 90% or more was obtained even for 25 PDL. Meanwhile,in the CHO cells retaining HAC vector (21HAC2) carrying GFP derived fromchromosome 21 described by Kazuki et al. (Gene Therapy: PMID: 21085194,2010), the retention rate was 70% or less for 25 PDL. The representativeresults are given in FIG. 34.

[A. 2] Stability of the Mouse Artificial Chromosome Vector MAC1 in MouseES Cell

Under non-selection culture of 0 to 75 PDL for the mouse ES clones (forexample, KO56 (MAC1)-5 and TT2F (MAC1)-23, obtained from Example 2above) obtained from the above, the rate of cells retaining MAC1 afterlong-term culture was measured by FISH analysis. As a result, theretention rate of 90% or more was obtained even for 75 PDL. On the otherhand, in the mouse ES cells retaining HAC vector (21HAC2) carrying GFPderived from chromosome 21 described by Kazuki et al. (Gene Therapy:PMID: 21085194, 2010), the retention rate was 70% or less for 75 PDL.The representative results are given in FIG. 35.

[A. 3] Preparation of Chimeric Mouse Retaining Artificial ChromosomeVector MAC1

By using the ES cell clones obtained from Example 2 above, a chimericmouse was prepared according to the method described by Tomizuka et al.(Nature Genet. 16: 133, 1997). As a host cell, an eight-cell stageembryo obtained by sexual crossbreeding of MCH (ICR) (white, purchasedfrom CLEA Japan, Inc.) was used. Injected embryo is transplanted into afoster mother, and coat color of the new-born mouse could be examined inorder to see whether or not it is a chimera. As the result that 1620embryos injected with ES clones retaining MAC1 (for example, KO56MAC1-5and TT2FMAC1-4, obtained from Example 2 above) were transplanted intofoster mothers, 56 chimeric mice (in which dark brown color area wasobserved in coat color) were born. Among them, 13 animals wereindividuals having a chimeric rate of about 100% from which almost nowhite color area was observed. In other words, it was shown that ES cellline (KO56 and TT2F) retaining the mouse artificial chromosome vectorMAC1 retained a chimera forming ability, that is, an ability ofdifferentiating into normal tissue of a mouse individual.

[A. 4] Transmission to Progeny of MAC1 from Chimeric Mouse Retaining theMouse Artificial Chromosome Vector MAC1

Two female chimeric mice (chimeric rate: about 100%) prepared from theabove [A. 3] were mated with male mice MCH (ICR) (white, purchased fromCLEA Japan, Inc.). Among 18 new-born mice born from chimeric mice, 13were dark brown color, which indicated the retention of a dominantgenetic trait derived from the ES cells. Thus, the ES cell lineretaining MAC1 was proven to be differentiated into a functional eggcell in a female chimeric mouse. Further, the retention of MAC1 wasexamined based on GFP fluorescence. As a result, six animals out of the13 (46%) were GFP positive and thus it was confirmed that the progeny ofthe chimeric mice have retained MAC1. Specifically, according to theMendel's genetics law, it was confirmed that the MAC1 trait appearedwith frequency of about 50%, and therefore it was shown that theretention rate of MAC1 is close to 100% in an ovum. The mouse lineage inwhich MAC1 was transmitted to progeny is referred to as TC (MAC1).

[A.5] Stability of MAC1 in Somatic Cells of TC (MAC1) Mouse Lineage

[A. 5. 1] Observation with Stereo Fluorescence Microscope

For each one of the male (5) and female (2) TC (MAC1) mice obtained fromthe above, the brain, thymus, heart, lung, liver, kidney, spleen, smallintestine, muscle, and testis (or ovary) were observed under stereofluorescence microscope. As a result, all tissues were observed to beGFP positive, and therefore the positive rate was 100%. Representativeresults of the female (5) are given in FIG. 36.

[A. 5. 2] FACS Analysis of Hematopoietic Cells

By using an antibody (Becton, Dickinson and Company) specific for Bcells (CD19), T cells (CD4 and CD8), and megakaryocyte (CD41), GFPpositive rate was studied for bone marrow and spleen cells. As a result,the positive rate was 95% or more in all tissues. On the other hand, inthe mouse retaining HAC vector (21HAC2) carrying GFP derived fromchromosome 21 described by Kazuki et al. (Gene Therapy: PMID: 21085194,2010), the positive rate was 15% or less in all tissues. Therepresentative results are given in FIG. 37 and FIG. 38.

[A. 5. 3] Fluorescence In Situ Hybridization (FISH) Analysis

By using the tail fibroblast prepared from the same individual as above,FISH analysis was carried out by using mouse minor satellite DNA as aprobe according to the method described by Shinohara et al. (HumanMolecular Genetics, 10: 1163-1175, 2001). As a result, the presence ofMAC1 was visually confirmed and it was confirmed that MAC1 was presentseparate from the mouse chromosome in 95% or more cells (FIG. 39).

From these results, it was confirmed that the mouse artificialchromosome vector MAC1 was very stably maintained at a rate of 90% ormore in the mouse ES cells (in vitro) and mouse tissues (in vivo).

Example 8 Preparation and Stability of Mouse Retaining the MouseArtificial Chromosome Vector CYP3A-MAC

[A] Transfer of CYP3A-MAC from CHO Cell to Mouse A9 Cell

To prepare mouse ES cells retaining CYP3A-MAC, introduction was carriedout from CHO cells (CHO (CYP3A-MAC, hChr7-ΔCYP3A) 22, 26, 34, 35, or thelike) retaining CYP3A-MAC obtained from Example 3 above, to, as a mouseA9 cell, mouse A9 cells having high microcell forming ability bymicrocell fusion. Total 25 resistant colonies obtained by eightmicrocell fusions were isolated, amplified, and subjected to thefollowing analysis (clone name: A9 (CYP3A-MAC)). As a result, there weresix clones which were determined to be positive by PCR using the primersdescribed above for detecting the CYP3A-MAC region only. In addition,FISH analysis (Tomizuka et al., Nature Genet. 16: 133, 1997) was carriedout by using CYP3A-BAC(RP11757A13) (CHORI) and mouse minor satellite DNAas probes, and as a result, the presence of CYP3A-MAC, which wasspecifically detected with the probes, was confirmed in three clones outof the six clones (FIG. 40). From the above results, it was concludedthat three clones of A9 cells retaining CYP3A-MAC were obtained.

[B] Transfer of CYP3A-MAC from A9 Cell to Mouse ES Cell

To prepare a chimeric mouse retaining CYP3A-MAC, introduction wascarried out from A9 cells retaining CYP3A-MAC obtained from the above[A] to mouse ES cells (wild type TT2F) by microcell fusion. According tothe method of Tomizuka et al. (Nature Genet. 16: 133, 1997), microcellswere purified from approximately 10⁸ A9 cells retaining CYP3A-MAC (A9(CYP3A-MAC) 8, 9, or the like) and suspended in 5 ml of DMEM.Approximately 10⁷ mouse ES cells of TT2F were detached by trypsintreatment, washed three times with DMEM, suspended in 5 ml of DMEM, andadded to microcells obtained by centrifugation. After centrifugation for10 min at 1250 rpm, the supernatant was completely removed. Theprecipitates were resolved fully by tapping, added with 0.5 ml of 1:1.4PEG solution [5 g of PEG1000 (Wako Pure Chemical Industries, Ltd.) and 1ml of DMSO (Sigma) are dissolved in 6 ml of DMEM], and fully stirred forabout 1 min and 30 sec. After that, 10 ml of DMEM was slowly added,centrifuged for 10 min at 1250 rpm, and suspended in 30 ml of ES culturemedium. Thereafter, the cells were dispensed into three petri disheswith a diameter of 100 mm (Corning Incorporated) onto which feeder cellswere previously plated and then cultured. 24 hours later, the culturemedium was exchanged with culture medium containing 300 μg/ml of G418and then subjected to selection culture for about 1 week. As a result,total 34 colonies were isolated, amplified, and subjected to thefollowing analysis. 14 clones from A9 (CYP3A-MAC) 8 and seven clonesfrom A9 (CYP3A-MAC) 9 were determined to be positive by PCR using theprimers described above for detecting the CYP3A-MAC region only. Inaddition, for 20 clones among the above, FISH analysis (Tomizuka et al.,Nature Genet. 16: 133, 1997) was carried out by using DNA derived fromCYP3A-BAC(RP11-757A13) (CHORI). As a result, the clones that werespecifically detected with the probes and had normal mouse nuclear type,were found to be eight clones (FIG. 41). From the above results, it wasconcluded that eight clones of TT2F cells retaining CYP3A-MAC wereobtained.

[C] Stability of CYP3A-MAC in Mouse ES Cell

Under non-selection culture of 0 to 100 PDL for the mouse ES clonesobtained from the above (for example, TT2F (CYP3A-MAC) 8-5, 8-22, 9-4,9-7, and 9-9, obtained from the above [B]), the rate of cells retainingCYP3A-MAC after long-term culture was measured by FISH analysis. As aresult, the retention rate of 95% or more was obtained even for 100 PDL(FIG. 42).

[D] Preparation of Chimeric Mouse Retaining CYP3A-MAC

By using the ES cell clones retaining CYP3A-MAC obtained from the above[B], chimeric mice were prepared according to the method of Tomizuka etal. (Nature Genet. 16: 133, 1997). As a host cell, eight-cell stageembryos obtained by sexual crossbreeding of MCH (ICR) (white, purchasedfrom CLEA Japan, Inc.) were used. Injected embryo was transplanted intoa foster mother, and coat color of the new-born mouse was examined tosee whether or not it is a chimera. As the result that 840 embryosinjected with ES clones retaining MAC1 (for example, TT2F (CYP3A-MAC)8-5, 8-16, 8-22, 9-4, 9-7, 9-9, 9-10, or the like, obtained from theabove [B]) were transplanted into a foster mother, 28 chimeric mice (inwhich dark brown color area was observed in coat color) were born. Amongthem, five were individuals having a chimeric rate of about 100% fromwhich almost no white area was observed. In other words, it was shownthat ES cell line (TT2F) retaining the mouse artificial chromosomevector CYP3A-MAC retains a chimera forming ability, that is, an abilityof differentiating into normal tissue of a mouse individual.

[E] Transmission to Progeny of CYP3A-MAC from Chimeric Mouse RetainingCYP3A-MAC

Five female chimeric mice (chimeric rate: about 100%) prepared from theabove [D] were mated with male mice MCH (ICR) (white, purchased fromCLEA Japan, Inc.). Among 60 new-born mice born from a chimeric mouse, 50were dark brown color, which indicated retention of a dominant genetictrait derived from the ES cells. Thus, the ES cell line retainingCYP3A-MAC was proven to be differentiated into a functional egg cell ina female chimeric mouse. Further, the retention of CYP3A-MAC wasexamined based on GFP fluorescence. As a result, 29 animals out of the50 (58%) were found to be GFP positive and it was confirmed thatCYP3A-MAC was retained in the progenies of a chimeric mouse.Specifically, according to the Mendel's genetics law, it was confirmedthat CYP3A-MAC trait appeared with frequency of about 50%, and thereforethe retention rate of CYP3A-MAC was close to 100% in an ovum. The mouselineage in which CYP3A-MAC was transmitted to a progeny is referred toas TC(CYP3A-MAC).

[F] Stability of CYP3A-MAC in Somatic Cells of TC(CYP3A-MAC) MouseLineage

[F. 1] Observation with Stereo Fluorescence Microscope

For each one of the male (2) and female (14) TC(CYP3A-MAC) mice obtainedfrom the above, the brain, thymus, heart, lung, liver, kidney, spleen,small intestine, muscle, and testis were observed under stereofluorescence microscope. As a result, all tissues were observed to beGFP positive, and the positive rate was 100%. Representative results ofthe male (2) are given in FIG. 43.

[F. 2] FACS Analysis of Hematopoietic Cells

By using an antibody (Becton, Dickinson and Company) specific for Bcells (CD19), T cells (CD4 and CD8), and megakaryocyte (CD41), GFPpositive rate was examined for bone marrow. As a result, the positiverate was 94% or more in all tissues. In contrast, in the mouse retainingHAC vector (CYP3A-HACΔ) derived from chromosome 14 described in WO2009/063722 (PCT/JP2008/068928), the positive rate was 20% or less inall tissues. The representative results are given in FIG. 44.

[F. 3] Fluorescence In Situ Hybridization (FISH) Analysis

For the same individual or tissues as described above, FISH analysis wascarried out by using CYP3A-BAC(RP11-757A13) DNA as a probe according tothe method described by Shinohara et al. (Human Molecular Genetics, 10:1163-1175, 2001). As a result, the presence of CYP3A-MAC was visuallyconfirmed and it was confirmed that CYP3A-MAC was present in 90 to 98%or more of the cells. In contrast, in the mouse retaining HAC vector(CYP3A-HACΔ) derived from chromosome 14 described in WO 2009/063722(PCT/JP2008/068928), the positive rate was 56 to 97% in all tissues. Therepresentative results are given in FIG. 45.

[F. 4] Transmission Rate of TC(CYP3A-MAC) Line

Eight TC(CYP3A-MAC) female mice were mated with eight male mice MCH(ICR) (white, purchased from CLEA Japan, Inc.) to examine the transferrate. Among 81 new-born mice obtained, 38 animals were GFP negativeindividuals and 42 animals were GFP positive individuals (transmissionrate: 53%). Thus, the transmission rate matched the Mendel's geneticslaw, and it was confirmed that CYP3A-MAC trait appeared with frequencyof about 50%, and therefore it was shown that the retention rate ofCYP3A-MAC was close to 100% in an ovum.

[F. 5] Preparation of TC(CYP3A-MAC) Homozygous Line Retaining TwoCYP3A-MAC and Transmission Rate

The TC(CYP3A-MAC) male mouse and TC(CYP3A-MAC) female mouse obtainedfrom the above were mated with each other to attempt the establishmentof TC(CYP3A-MAC) homozygous line retaining two CYP3A-MAC. By using tailfibroblast of 18 new-born mice, FISH analysis was carried out by usingCYP3A-BAC(RP11757A13) DNA as a probe according to the method describedby Shinohara et al. (Human Molecular Genetics, 10: 1163-1175, 2001). Asa result, the presence of CYP3A-MAC was visually confirmed. In 4×36lines, three had two copies, five had one copy, and four had zero copyamong 12 new-born mice. In 24×37 lines, one had two copies, three hadone copy, and two had zero copy among six new-born mice. Total 18animals were obtained, and the rate of CYP3A-MAC having two copies (fouranimals), one copy (eight animals), and zero copy (six animals) was1:2:1.5, which almost matched the Mendel's genetics law. Therepresentative results are given in FIG. 46.

From these results, it was confirmed that CYP3A-MAC was very stablymaintained for a long period of time at a rate of 95% or more in themouse ES cells (in vitro) and, as a homozygous line, it was also verystably maintained at a rate of 90% or more in the mouse tissues (invivo).

[G] Tissue Specific Gene Expression of CYP3A Gene Cluster inTC(CYP3A-MAC) Mouse Lines

For each one of the male (2) and female (14) TC(CYP3A-MAC) mice obtainedfrom the above, total RNA was extracted from the brain, thymus, heart,lung, liver, kidney, spleen, small intestine, and muscle of the animalsaccording to the commercially available protocol (QIAGEN), and cDNA wassynthesized according to the commercially available protocol(invitrogen). Consequently, PCR was performed by using the cDNA as atemplate and expression of human CYP3A gene cluster and mouse Cyp3a genecluster was detected. The primer sequences are given below.

Primers for detecting expression of human CYP3A gene cluster are:

3A4-1L: (SEQ ID NO: 51) 5′-gtatggaaaagtgtggggct-3′ 3A4-1R:(SEQ ID NO: 52) 5′-atacttcaagaattgggatg-3′ 3A4-2L: (SEQ ID NO: 53)5′-ccaagctatgctcttcaccg-3′ 3A4-2R: (SEQ ID NO: 54)5′-tgaagaagtcctcctaagct-3′ 3A5-1L: (SEQ ID NO: 55)5′-ctctgtttccaaaagatacc-3′ 3A5-1R: (SEQ ID NO: 56)5′-tcaacatctttcttgcaagt-3′ 3A7-1L: (SEQ ID NO: 57)5′-agcttttaagatttaatcca-3′ 3A7-1R: (SEQ ID NO: 58)5′-gagctttgtgggtctcagag-3′ 3A7-2L: (SEQ ID NO: 59)5′-ctctcagaattcaaaagact-3′ 3A7-2R: (SEQ ID NO: 60)5′-agaagaagtcctccaaagcg-3′ 3A43-2L: (SEQ ID NO: 61)5′-tatgacacaactagcaccac-3′ 3A43-2R: (SEQ ID NO: 62)5′-agtgtctagtgttctgggat-3′Primers for detecting expression of mouse Cyp3a gene cluster are:

3a11-1L: (SEQ ID NO: 63) 5′-tcaaacgcctctccttgctg-3′ 3a11-1R:(SEQ ID NO: 64) 5′-gcttgcctttctttgccttc-3′ 3a11-2L: (SEQ ID NO: 65)5′-ggtaaagtacttgaggcaga-3′ 3a11-2R: (SEQ ID NO: 66)5′-agaaagggctttatgagaga-3′ 3a13-1L: (SEQ ID NO: 67)5′-agaaacatgaggcagggatt-3′ 3a13-1R: (SEQ ID NO: 68)5′-acaaggagacatttagtgca-3′ 3a13-2L: (SEQ ID NO: 69)5′-taccccagtatttgatgcac-3′ 3a13-2R: (SEQ ID NO: 70)5′-agataactgactgagccaca-3′Primers for detecting expression of control gene are:

GAPDH-F: (SEQ ID NO: 71) 5′-CCATCTTCCAGGAGCGAGA-3′ GAPDH-R:(SEQ ID NO: 72) 5′-TGTCATACCAGGAAATGAGC-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and EX Taq (TAKARA SHUZO CO., LTD.) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 93° C. for 5 min,35 cycles of 93° C. for 1 min, 56° C. for 1 min, and 72° C. for 1 min asone cycle were carried out.

As a result, in the mouse retaining TC(CYP3A-MAC), expression of CYP3A4was able to be detected only in the liver and small intestine,expression of CYP3A5 was able to be detected only in the liver, smallintestine, and lung, expression of CYP3A7 was able to be detected onlyin the liver, small intestine, kidney, and lung, expression of CYP3A43was able to be detected only in the liver, small intestine, and kidney,expression of Cyp3a11 was able to be detected only in the liver andsmall intestine, and expression of Cyp3a13 was able to be detected onlyin the liver and small intestine. On the other hand, GAPDH as a controlwas detected in all tissues. The representative results of the female(14) are given in FIG. 47. Tissue specific expression was thus observedas seen in human, indicating the humanization.

[H] Time Specific Gene Expression of CYP3A Gene Cluster in TC(CYP3A-MAC)Mouse Lines

From the male and female TC(CYP3A-MAC) mice which were GFP positive,total RNA was extracted from the liver at the fetal age of 14.5 days,fetal age of 16.5 days, fetal age of 18.5 days, day 0 after birth, 4weeks old, 6 weeks old, 12 weeks old, and 24 weeks old according to thecommercially available protocol (QIAGEN) and cDNA was synthesizedaccording to the commercially available protocol (invitrogen). PCR wasperformed by using the cDNA as a template and expression was detected byusing the primers for detecting the expression of human CYP3A genecluster and mouse Cyp3a gene cluster described above.

As a result, it was confirmed that the adult-expression type humanCYP3A4, human CYP3A5, mouse cyp3a11, and mouse Cyp3a13 are stronglyexpressed in an adult period and the fetal type CYP3A7 was stronglyexpressed in a fetal. In addition, the expression level of GAPDH as acontrol was detected equally in all animals of any fetal age or weekage. The representative results are given in FIG. 48. Time specificexpression was thus observed as in human, indicating the humanization.

Example 9 Preparation of TC(CYP3A-MAC)/Δcyp Mouse Lines

[A] Construction of Mouse Lines Retaining CYP3A-MAC and Having DisruptedTwo Alleles of Endogenous Cyp3a Gene Group

TC(CYP3A-MAC) prepared in Example 8 above was subjected to back crosswith Δcyp line that was prepared in Example 7 of WO 2009/063722(PCT/JP2008/068928) to yield GFP-positive mouse individuals. Genotypeanalysis was performed for the mice using the PCR method describedabove. Tail of the 51 new-born mice obtained by crossbreeding waspartially cut off, and genomic DNA was prepared from the tail sample.For the obtained DNA, PCR was carried out by using the primers fordetecting CYP3A-MAC and the primers described in Table 1 of WO2009/063722 (PCT/JP2008/068928) in the same manner as above in order toexamine the retention of CYP3A-MAC and the KO of Cyp3a gene cluster. Asa result, it was confirmed that 24 animals were (heterozygous KO) mouselines in which CYP3A-MAC was retained and one allele of the endogenousCyp3a gene cluster was disrupted. Further, the mouse in which CYP3A-MACwas retained and a group of Cyp3a genes was heterozygously disrupted,was subjected to back cross with Δcyp line. Tail of the 38 GFP-positivemice obtained was partially cut off, and genomic DNA was prepared fromthe tail sample. Genotype analysis was performed by the same PCR methodas described above. As a result, it was confirmed that 18 animals were(homozygous KO) mouse lines in which CYP3A-MAC was retained and bothalleles of the endogenous Cyp3a gene group were disrupted (hereinbelow,described as TC(CYP3A-MAC)/Δcyp).

[B] Metabolism Analysis of TC(CYP3A-MAC)/Δcyp Mouse Line

According to Omura et al. (J. Biol. Chem., 239, 2370, 1964), the livermicrosome of TC(CYP3A-MAC)/Δcyp mouse and Δcyp mouse individual is mixedwith triazolam (200 μM), which is known to be metabolized into CYP3A4.As a result, α-OH-triazolam and 4-OH-triazolam can be measured asmetabolites. As described in WO 2009/063722 (PCT/JP2008/068928), inTC(CYP3A-MACΔ)/Δcyp mouse, it is possible to confirm that it has thesame activity as the mouse of the same line or human (HLM: human livermicrosome). From the above, it is able to confirm that, inTC(CYP3A-MAC)/Δcyp mouse line, the human CYP3A gene on CYP3A-MAC isfunctional and also equivalent to the human gene.

[C] Thus, the liver microsome derived from TC(CYP3A-MAC)/Δcyp mouse linecan be used as a sample for testing a pharmacological effect andtoxicity in the phase I reaction for development of a pharmaceuticalproduct. Further, because human drug metabolism can be reproduced inTC(CYP3A-MAC)/Δcyp mouse line, it can be also used as a model mouse forin vivo test that is used for testing a pharmacological effect andtoxicity in the phase I reaction for development of a pharmaceuticalproduct.

Example 10 Preparation of Rat Retaining the Mouse Artificial ChromosomeVector CYP3A-MAC

[A] Transfer of CYP3A-MAC from A9 Cell to Rat ES Cell

To prepare a chimeric rat retaining CYP3A-MAC, introduction was carriedout from A9 cells retaining CYP3A-MAC obtained from Example 8 above torESWIv3i-1 (Hirabayashi et al., Mol Reprod Dev. 2010 February; 77 (2):94), which was a rat ES cell capable of transmission to progeny, bymicrocell fusion. According to the method of Tomizuka et al. (NatureGenet. 16: 133, 1997), microcells were purified from approximately 10⁸A9 cells retaining CYP3A-MAC (A9 (CYP3A-MAC) 8, 9, or the like) andsuspended in 5 ml of DMEM. Approximately 10⁷ rat ES cells of rESWIv31-1were detached by trypsin treatment, washed three times with DMEM,suspended in 5 ml of DMEM, and added to the microcells obtained bycentrifugation. After centrifugation for 10 min at 1250 rpm, thesupernatant was completely removed. The precipitates were resolved fullyby tapping and added with 0.5 ml of 1:1.4 PEG solution [5 g of PEG1000(Wako Pure Chemical Industries, Ltd.), and 1 ml of DMSO (Sigma) aredissolved in 6 ml of DMEM], and fully stirred for about 1 min and 30sec. After that, 10 ml of DMEM was slowly added, centrifuged for 10 minat 1250 rpm, and suspended in 30 ml of ES culture medium. Thereafter,the cells were dispensed into three petri dishes with a diameter of 100mm (Corning Incorporated) onto which feeder cells were previously platedand then cultured. 24 hours later, the culture medium was exchanged withculture medium containing 300 μg/ml G418 and then subjected to selectionculture for about 1 week. As a result, total 10 colonies were isolated,amplified, and subjected to the following analysis. Two clones from A9(CYP3A-MAC) 8 and three clones from A9 (CYP3A-MAC) 8 were determined tobe positive by PCR using the primers described above for detecting theCYP3A-MAC region only. In addition, for the five clones, FISH analysis(Tomizuka et al., Nature Genet. 16: 133, 1997) was carried out by usingCYP3A-BAC (RP11-757A13) (CHORI) and mouse Cot-1 DNA. As a result, theclones that were specifically detected with the probes and had normalrat nuclear type, were found to be three clones (FIG. 49). From theabove, it was concluded that 3 clones of rat ES cells retainingCYP3A-MAC were obtained.

[B] As described in Example 8, in vitro stability can be examined byusing rat ES cells retaining the mouse artificial chromosome vectorCYP3A-MAC. Further, by preparing a chimeric rat using the ES cells, ratline rTC(CYP3A-MAC) in which the vector has been transmitted to a ratprogeny can be prepared. Further, by using the rTC(CYP3A-MAC) rat line,stability of CYP3A-MAC in somatic cells can be examined. Still further,the liver microsome derived from rTC(CYP3A-MAC) rat line can be used asa sample for testing a pharmacological effect and toxicity in the phaseI reaction for development of a pharmaceutical product. Further, becausehuman drug metabolism can be reproduced in rTC (CYP3A-MAC) rat line, itcan be also used as a model rat for in vivo test that is used fortesting a pharmacological effect and toxicity in the phase I reactionfor development of a pharmaceutical product.

Example 11 Construction of the Mouse Artificial Chromosome VectorhChr21q-MAC

In order to prepare a model mouse for Down's syndrome, translocationcloning of DNA sequence containing 33 Mb region distal from AP001657 oflong arm of human chromosome 21 into the mouse artificial chromosomevector MAC1 is performed by using Cre/loxP system to constructhChr21q-MAC in the same manner as in Example 3.

[A] Introduction of hChr21-loxP from DT40 Containing hChr21-loxP to CHOCell Containing MAC1

For translocation insertion of a region distal from AP001657 of long armof human chromosome 21 into the mouse artificial chromosome vector MAC1via loxP sequence in CHO cells, hChr21-loxP which is obtained byinserting loxP sequence into AP001657 in human chromosome 21 isintroduced into CHO cells containing the mouse artificial chromosomevector MAC1.

[A. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 cells containing hChr21-loxP and DT40 (kk139) (JapanesePatent Publication (Kokai) 2007-295860 A) as a recipient cell, microcellfusion was carried out for CHO(HPRT⁻; MAC1), which is CHO hprt depletedcells containing MAC1 (obtained from the Health Science ResearchResources Bank, registration number: JCRB0218), in the same manner asabove. Total 114 resistant colonies obtained by 14 microcell fusionswere isolated, amplified, and subjected to the following analysis (clonename: CHO (HPRT⁻; MAC1, hChr21-loxP)).

[A. 2] Selection of Drug Resistant Clone

[A. 2. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out for 60clones out of the 114 clones by using the following primers, and it wasconfirmed whether or not human chromosome 21 fragment has beenintroduced into the CHO cells retaining MAC1. The primer sequences aregiven below.

m11 5L (described above) EGFP (F) L (described above)kj neo (described above) m11 6R (described above) #21CEN<1>2L:(SEQ ID NO: 73) 5′-aaatgcatcaccattctcccagttaccc-3′ PGKr1:(SEQ ID NO: 74) 5′-ggagatgaggaagaggagaaca-3′ D21S265-L: (SEQ ID NO: 75)5′-gggtaagaaggtgcttaatgctc-3′ D21S265-R: (SEQ ID NO: 76)5′-tgaatatgggttctggatgtagtg-3′ D21S261-L: (SEQ ID NO: 77)5′-gagggggactgggacaagccctttgctggaagaga-3′ D21S261-R: (SEQ ID NO: 78)5′-acattaggaaaaatcaaaaggtccaattattaagg-3′ D21S268-L: (SEQ ID NO: 79)5′-CAACAGAGTGAGACAGGCTC-3′ D21S268-R: (SEQ ID NO: 80)5′-TTCCAGGAACCACTACACTG-3′ D21S266-L: (SEQ ID NO: 81)5′-ggcttggggacattgagtcatcacaatgtagatgt-3′ D21S266-R: (SEQ ID NO: 82)5′-gaagaaaggcaaatgaagacctgaacatgtaagtt-3′ D21S1259-L: (SEQ ID NO: 83)5′-GGGACTGTAATAAATATTCTGTTGG-3′ D21S1259-R: (SEQ ID NO: 84)5′-CACTGGCTCTCCTGACC-3′ CBR-L: (SEQ ID NO: 85) 5′-gatcctcctgaatgcctg-3′CBR-R: (SEQ ID NO: 86) 5′-gtaaatgccctttggacc-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,11 clones out of the 60 clones were found to be positive for all primersets, and the following analysis was performed by using those 11 clones.

[A. 2. 2] Two-Color FISH Analysis

With six clones out of the 11 clones of CHO(HPRT⁻; MAC1, hChr21-loxP)obtained from the above, FISH analysis was carried out by using mouseCot-1 DNA and human Cot-1 DNA as probes according to the methoddescribed by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175,2001). As a result, it was confirmed that a single copy of MAC1 andhChr21-loxP has introduced into CHO cells at a rate of 70% in one cloneout of the six clones (FIG. 50).

From these results, it was concluded that hChr21-loxP could beintroduced into CHO cells containing the mouse artificial chromosomevector MAC1.

[B] Site Specific Translocation of 33 Mb Region Distal from AP001657 ofLong Arm of Human Chromosome 21 into MAC1 Vector in CHO(HPRT⁻; MAC1,hChr21-loxP) Clone

To stably keep a region distal from AP001657 of long arm of humanchromosome 21, which is a DNA having 33 Mb size, in a mouse individual,translocation insertion into the mouse artificial chromosome vector MAC1was performed (FIG. 51).

[B. 1] Transfection and Isolation of HAT Resistant Clone

Gene transfer was carried out by lipofection for the CHO(HPRT⁻; MAC1,hChr2′-loxP)-37 obtained from the above. To cells in 6 wells with 90%confluency, 3 μg of Cre was added according to the commerciallyavailable protocol (Invitrogen). After 2-weeks culture conducted underHAT selection culture, a resistant colony was generated and total twocolonies obtained by two gene transfers were isolated, amplified, andsubjected to the following analysis (clone name: CHO (hChr21q-MAC,hChr2′-hChr21q)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from HAT resistant cell line and using it asa template for selecting a clone with reciprocal translocation, PCR wascarried out by using the following primers and it was confirmed whetheror not reciprocal chromosomal translocation has occurred on humanchromosome 21 fragment and MAC1. The primer sequences are given below.

kj neo (described above)

PGKr1 (described above)

D215265-L (described above)

D21S265-R (described above)

D21S261-L (described above)

D21S261-R (described above)

D215268-L (described above)

D21S268-R (described above)

D21S266-L (described above)

D21S266-R (described above)

D21S1259-L (described above)

D21S1259-R (described above)

CBR-L (described above)

CBR-R (described above)

TRANS L1 (described above)

TRANS R1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,all two clones were found to be positive for all primer sets and thefollowing analysis was performed by using those two clones.

[B. 2. 2] Two-Color FISH Analysis

With the two clones of CHO (hChr21q-MAC, hChr21-hChr21q) obtained fromthe above, FISH analysis was carried out by using mouse Cot-1 DNA andhuman Cot-1 DNA as probes according to the method described by Shinoharaet al. (Human Molecular Genetics, 10: 1163-1175, 2001). As a result, itwas confirmed that the signal derived from human chromosome 21 wasobserved on MAC1 at a rate of 90% or more in two clones out of the twoclones (FIG. 52).

From these results, it was concluded that cloning of 33 Mb region distalfrom AP001657 of long arm of human chromosome 21 into the mouseartificial chromosome vector MAC1 could be achieved by reciprocaltranslocation.

[C] Transfer of hChr21q-MAC from CHO Cell to Mouse ES Cell

To prepare a chimeric mouse retaining hChr21q-MAC, transfer was carriedout from CHO cells retaining hChr21q-MAC obtained from the above [B] tomouse ES cells (wild type TT2F) by microcell fusion. According to themethod of Tomizuka et al. (Nature Genet. 16: 133, 1997), microcells werepurified from approximately 10⁸ cells of CHO retaining hChr21q-MAC(CHO(hChr21q-MAC, hChr21-hChr21q) 1, 2)) and suspended in 5 ml of DMEM.Approximately 10⁷ mouse ES cells TT2F were detached by trypsintreatment, washed three times with DMEM, suspended in 5 ml of DMEM, andadded to the microcells obtained by centrifugation. After centrifugationfor 10 min at 1250 rpm, the supernatant was completely removed. Theprecipitates were resolved fully by tapping and added with 0.5 ml of1:1.4 PEG solution [5 g of PEG1000 (Wako Pure Chemical Industries, Ltd.)and 1 ml of DMSO (Sigma) are dissolved in 6 ml of DMEM], and fullystirred for about 1 min and 30 sec. After that, 10 ml of DMEM was slowlyadded, centrifuged for 10 min at 1250 rpm, and suspended in 30 ml of ESculture medium. Thereafter, the cells were dispensed into three petridishes with a diameter of 100 mm (Corning Incorporated) to which feedercells have been previously added and then cultured. 24 hours later, theculture medium was exchanged with culture medium containing 300 μg/mlG418 and then subjected to selection culture for about 1 week. As aresult, total 24 colonies were isolated, amplified, and subjected to thefollowing analysis. Two clones from CHO (hChr21q-MAC, hChr2′-hChr21q) 1and six clones from CHO (hChr21q-MAC, hChr2′-hChr21q) 2 were determinedto be positive by PCR using the primers described before for detectingthe hChr21q-MAC region only. In addition, for eight clones among theabove, FISH analysis (Tomizuka et al., Nature Genet. 16: 133, 1997) wascarried out by using human Cot-1 DNA and mouse minor satellite DNA. As aresult, the clones that were specifically detected with the probes andhad normal mouse nuclear type were found to be two clones (FIG. 53).From the above, it was concluded that two clones of TT2F cells retaininghChr21q-MAC were obtained.

[D] Stability of hChr21q-MAC in Mouse ES Cell

Under non-selection culture of 0 to 50 PDL for the mouse ES clonesobtained from the above (for example, TT2F (hChr21q-MAC) 22, obtainedfrom the above [C]), the rate of cells retaining hChr21q-MAC afterlong-term culture was measured by FISH analysis. As a result, theretention rate of 95% or more was obtained even for 50 PDL (FIG. 54).

[E] Preparation of Chimeric Mouse Retaining hChr21q-MAC

By using the ES cell clones retaining hChr21q-MAC obtained from theabove [C], a chimeric mouse was prepared according to the method ofTomizuka et al. (Nature Genet. 16: 133, 1997). As a host cell,eight-cell stage embryos obtained by sexual crossbreeding of MCH (ICR)(white, purchased from CLEA Japan, Inc.) were used. Injected embryo wastransplanted into a foster mother, and coat color of the new born mousewas examined to see whether or not it is a chimera. As the result that220 embryos injected with ES clone retaining MAC1 (for example, TT2F(hChr21q-MAC) 20, 22, or the like obtained from the above [C]) weretransplanted into foster mothers, 18 chimeric mice (in which dark browncolor area was observed in coat color) were born. Among them, twoanimals were individuals having a chimeric rate of about 100% from whichalmost no white color area was observed. Further, one of them was aGFP-positive individual (FIG. 55). In other words, it was shown that EScell line (TT2F) retaining the mouse artificial chromosome vectorhChr21q-MAC retained a chimera forming ability, that is, an ability ofdifferentiating into normal tissue of a mouse individual.

[F] As described in Example 8, mouse line-based TC (hChr21q-MAC) inwhich hChr21q-MAC is transmitted to a progeny can be prepared from thechimeric mouse retaining the mouse artificial chromosome vectorhChr21q-MAC. Further, by using the TC (hChr21q-MAC) mouse line,stability of hChr21q-MAC in somatic cells can be examined. Stillfurther, the TC (hChr21q-MAC) line can be used as a model mouse forDown's syndrome, and it can be advantageously used for elucidating themechanism for onset of Down's syndrome or developing a therapeutic agentfor alleviating the symptom.

Example 12 Construction of the Mouse Artificial Chromosome VectorhChr21q22.12-MAC

In order to prepare a mouse for also presenting Down's syndrome,translocation cloning of a DNA sequence containing a region distal fromAP00172 of long arm of human chromosome 21 into the mouse artificialchromosome vector MAC1 is performed by using Cre/loxP system toconstruct hChr21q22.12-MAC in the same manner as in Example 3.

[A] Site Specific Insertion of loxP Sequence into AP001721 in HumanChromosome 21

For translocation insertion into the mouse artificial chromosome vectorMAC1 via loxP sequence, loxP sequence is inserted into AP001721 proximalto DSCR (Down's syndrome critical region cluster) of human chromosome 21(hChr21) in DT40 cells.

[A. 1] Preparation of Targeting Vector pCKloxPHyg

Targeting vector pCKloxPHyg for inserting loxP, which is a recognitionsequence for Cre recombinase, into Down's syndrome causative gene region(DSCR), which is located extremely close to AP001721 of human chromosome21 and on the centromere side (i.e., locating on the centromere side byapproximately 50 Kb from AP001721) was prepared as follows. First, theAP001721 genome region was amplified by PCR using the following primers.

AML5′ .L1; (SEQ ID NO: 87) 5′-TAGAATTCGTAGGCTTGGAAGCAGTGAGAGAGAA-3′AML5′ .R2; (SEQ ID NO: 88) 5′-GAAGACTGGTAAATCTGGTGGCTGTC-3′ AML5′ .L4;(SEQ ID NO: 89) 5′-ATTAGATCTCCTGCTGTTATCTCATGCACTCTCA-3′ AML5′ .R4;(SEQ ID NO: 90) 5′-ATTAGATCTATGATGCCTGATACATGGTCTGTGA-3′

As a basic plasmid for inserting loxP sequence, V901 (Lexicon genetics)was used. For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. wasused as a thermal cycler and LA Taq (TAKARA SHUZO CO., LTD.) was used asTaq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used werethose included in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 5 min were carried out.PCR product was treated with proteinase K (Gibco) and the PCR fragments(2.9 kb and 2.0 kb) were subjected to gel filtration by usingCHROMASPIN-TE400 (Clontech). After that, the product was cleaved withthe restriction enzymes EcoRI (NIPPON GENE CO., LTD.) and BglII (NIPPONGENE CO., LTD.) and subjected to gel filtration by usingCHROMASPIN-TE1000 (Clontech). Thereafter, the MC1-TK sequence was cutout from V830 (Lexicon genetics) by using RsrII (NEB) and cloned intothe recognition site for restriction enzyme HindIII in V901 plasmid(V901T-1). The PCR fragments (2.9 kb and 2.0 kb) were cloned into theEcoRI and BglII sites of V901T-1 plasmid (V901T-1HR2). Next, by usingKpnI and AscI, the 5′-HPRT-loxP-Hyg was cut out from 5′-HPRT-loxP-Hyg-TKvector described by Kazuki et al. (Gene Therapy: PMID: 21085194, 2010),and then cloned into the AscI and KpnI sites of V901T-1HR2 (pCKloxPHyg).Size of the final construct inserted with loxP was 11.2 kb. Thetargeting vector, target sequence, and chromosome allele obtained byhomologous recombination are shown in FIG. 56.

[A. 2] Transfection and Isolation of Hygromycin Resistant Clone

As described above, the targeting vector pCKloxPHyg prepared above waslinearized with the restriction enzyme NotI (TAKARA), and used fortransfection of the DT40 hybrid cells retaining human chromosome 21(Kazuki et al. BBRC 2004, DT40 (21-2-3)). After exchanging the culturemedium for culture medium containing hygromycin B (1.5 mg/ml), the cellswere dispensed into three 96-well culture plates and then subjected toselection culture for about 2 weeks. Total 178 resistant coloniesobtained from four transfections were isolated, amplified, and subjectedto the following analysis (clone name: DT40 (hChr21q22.12-loxP)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

Genomic DNA was extracted from the hygromycin resistant clone by usingPuregene DNA Isolation Kit (Gentra Systems, Inc.) and identification ofthe homologous recombinant was carried out by PCR using the followingtwo sets of primers.

Identification of the homologous recombinant was carried out by PCRusing the following two sets of primers.

AMLloxP-4L; (SEQ ID NO: 91) 5′-AGAAAGGCAGGTGAGTGTGGAGGTAGA-3′AMLloxP-4R; (SEQ ID NO: 92) 5′-GAAGTGGGCTCACAGGAATTTTCCAA-3′ AMLloxP-8L;(SEQ ID NO: 93) 5′-GGGCCTCTTTATTTGGCAGAATATCACC-3′ AMLloxP-8R;(SEQ ID NO: 94) 5′-TTACACTGAGATTCAGGGCACGATGA-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 4 min were carried out. As a result ofscreening 178 clones, 71 clones were identified as a homologousrecombinant.

[A. 3. 2] Southern Blot Analysis

For the ten clones which have been confirmed to have recombination byPCR analysis above, Southern blot analysis was carried out as follows.The genomic DNA was treated with the restriction enzyme EcoRI (TAKARA),electrophoresed on 0.8% agarose gel, and subjected to alkali blottingusing a GeneScreen Plus™ hybridization transfer membrane (NENTM LifeScience Products, Inc.). The filter was then subjected to Southernhybridization by using SP7 probe, which has been obtained byamplification of the gene sequence in AP001721 by PCR, to identify thehomologous recombinant. For preparing SP7 probe, PCR was carried out byusing genomic DNA of DT40 (21-2-3) as a template and using the primersdescribed below, and ³²P labeled DNA probe was prepared by randompriming using the PCR product as a template (according to Amersham'sattached protocols).

Primers for preparing SP7 probe are:

SP7L; (SEQ ID NO: 95) 5′-CAGCTGGGAAACACTGAGCAAGATTATG-3′ SP7R;(SEQ ID NO: 96) 5′-CTGCTAGACTGAAAATGCGTTTCCTCTG-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and EX Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 93° C. for 5 min, 35 cycles of 93°C. for 1 min, 54° C. for 1 min, and 72° C. for 1 min as one cycle werecarried out. Based on Southern hybridization, it was expected that aband at approximately 7.5 kb was detected from the non-homologousrecombinant while a band at approximately 9.4 kb was detected from thehomologous recombinant (FIG. 56). As a result of Southern hybridization,it was found that all ten clones out of the ten clones were the desiredhomologous recombinant. The representative results are given in FIG. 57.

[A. 3. 3] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out forten clones out of the clones which have been confirmed to haverecombination in the above by using human cot-1 DNA and hygromycin asprobes. As a result, it was confirmed that human chromosome 21 was nottranslocated to the host chromosome in any clone, and based on the factthat hygromycin-derived signal was detected near 21q22, recombinationhas site-specifically occurred (FIG. 58). From these results, it wasconcluded that the loxP sequence as a gene introduction site wassite-specifically inserted into human chromosome 21 fragment.

[B] Transfer of hChr21q22.12-loxP from DT40 Containing hChr21q22.12-loxPto CHO Cell Containing MAC1

For translocation insertion of a region distal from AP001721 of long armof human chromosome 21 into the mouse artificial chromosome vector MAC1via loxP sequence in CHO cells, hChr21q22.12-loxP which was obtained byinserting loxP sequence into AP001721 in human chromosome 21 wasintroduced into CHO cells containing the mouse artificial chromosomevector MAC1.

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 cell containing hChr21q22.12-loxP, i.e. DT40(hChr21q22.12-loxP) 47, as a recipient cell, microcell fusion wascarried out for CHO(HPRT⁻; MAC1), that is, CHO hprt depleted cellscontaining MAC1 (obtained from the Health Science Research ResourcesBank, registration number: JCRB0218), in the same manner as above. Total140 resistant colonies obtained by 15 microcell fusions were isolated,amplified, and subjected to the following analysis (clone name:CHO(HPRT⁻; MAC1, hChr21q22.12-loxP)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out for 20clones out of the 140 clones above by using the following primers, andit was confirmed whether or not human chromosome 21 fragment has beenintroduced into the CHO cells containing MAC1. The primer sequences aregiven below.

m11 5L (described above) EGFP (F) L (described above)kj neo (described above) m11 6R (described above) D21S265-L:(SEQ ID NO: 97) gggtaagaaggtgcttaatgctc D21S265-R: (SEQ ID NO: 98)tgaatatgggttctggatgtagtg D21S261-L: (SEQ ID NO: 99)gagggggactgggacaagccctttgctggaagaga D21S261-R: (SEQ ID NO: 100)acattaggaaaaatcaaaaggtccaattattaagg D21S268-L: (SEQ ID NO: 101)CAACAGAGTGAGACAGGCTC D21S268-R: (SEQ ID NO: 102) TTCCAGGAACCACTACACTGD21S266-L: (SEQ ID NO: 103) ggcttggggacattgagtcatcacaatgtagatgtD21S266-R: (SEQ ID NO: 104) gaagaaaggcaaatgaagacctgaacatgtaagttD21S1259-L: (SEQ ID NO: 105) GGGACTGTAATAAATATTCTGTTGG D21S1259-R:(SEQ ID NO: 106) CACTGGCTCTCCTGACC CBR-L: (SEQ ID NO: 107)gatcctcctgaatgcctg CBR-R: (SEQ ID NO: 108) gtaaatgccctttggacc

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,13 clones out of the 20 clones were found to be positive for all primersets and the following analysis was performed by using those 13 clones.

[B. 2. 2] Two-Color FISH Analysis

With six clones out of the 13 clones of CHO(HPRT⁻; MAC1,hChr21q22.12-loxP) obtained from the above, FISH analysis was carriedout by using mouse Cot-1 DNA and human Cot-1 DNA as probes according tothe method described by Shinohara et al. (Human Molecular Genetics, 10:1163-1175, 2001). As a result, it was confirmed that singly copy of MAC1and hChr21q22.12-loxP have been introduced into CHO cells at a rate of75% in two clones out of the six clones (FIG. 59).

From these results, it was concluded that hChr21q22.12-loxP could beintroduced into CHO cells containing the mouse artificial chromosomevector MAC1.

[C] Site Specific Translocation of 12 Mb Region Distal from AP001721 ofLong Arm of Human Chromosome 21 in CHO(HPRT⁻; MAC1, hChr21q22.12-loxP)Clone

To stably keep a region distal from AP001721 of long arm of humanchromosome 21, which is a DNA having 12 Mb size, in a mouse individual,translocation insertion into the mouse artificial chromosome vector MAC1was performed (FIG. 60).

[C. 1] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection for the CHO(HPRT⁻;MAC1, hChr21q22.12-loxP)-12 and -13 obtained from the above. To cells in6 wells with 90% confluency, 3 μg of Cre was added according to thecommercially available protocol (Invitrogen). After culture for 2 weeksunder HAT selection culture, a resistant colony was generated and total19 colonies obtained by two introductions were isolated, amplified, andsubjected to the following analysis (clone name: CHO (hChr21q22.12-MAC,hChr2′-hChr21q22.12)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from HAT resistant cell line and using it asa template for selecting a clone with reciprocal translocation, PCR wascarried out by using the following primers and it was confirmed whetheror not reciprocal chromosomal translocation has occurred on humanchromosome 21 fragment and MAC1. The primer sequences are given below.

kj neo (described above)

PGKr1 (described above)

D215265-L (described above)

D21S265-R (described above)

D21 S261-L (described above)

D21S261-R (described above)

D215268-L (described above)

D215268-R (described above)

D215266-L (described above)

D215266-R (described above)

D21S1259-L (described above)

D21S1259-R (described above)

CBR-L (described above)

CBR-R (described above)

TRANS L1 (described above)

TRANS R1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,eight clones out of the 19 clones were found to be positive for allprimer sets, and the following analysis was performed by using those 8clones.

[C. 2. 2] Two-Color FISH Analysis

For 8 clones of CHO (hChr21q22.12-MAC, hChr2′-hChr21q22.12) obtainedabove, FISH analysis was carried out by using mouse Cot-1 DNA and humanCot-1 DNA as probes according to the method described by Shinohara etal. (Human Molecular Genetics, 10: 1163-1175, 2001). As a result, it wasconfirmed that the signal derived from human chromosome 21 was observedon MAC1 at a rate of 85% or more in all clones out of the eight clones(FIG. 61).

From these results, it was concluded that cloning of 12 Mb region distalfrom AP001721 of long arm of human chromosome 21 into the mouseartificial chromosome vector MAC1 could be achieved by reciprocaltranslocation.

[D] Transfer of hChr21q22.12-MAC from CHO Cell to Mouse ES Cell

To prepare a chimeric mouse retaining hChr21q22.12-MAC, transfer wascarried out from CHO cells retaining hChr21q22.12-MAC obtained from theabove [C] to mouse ES cells (wild type TT2F) by microcell fusion.According to the method of Tomizuka et al. (Nature Genet. 16: 133,1997), microcells were purified from approximately 10⁸ cells of CHOretaining hChr21 q-MAC(CHO (hChr21 q22. 12-MAC, hChr21-hChr21 q22. 1 2)1, 12, or the liked) and suspended in 5 ml of DMEM. Approximately 10⁷mouse ES cells of TT2F were detached by trypsin treatment, washed threetimes with DMEM, suspended in 5 ml of DMEM, and added to the microcellsobtained by centrifugation. After centrifugation for 10 min at 1250 rpm,the supernatant was completely removed. The precipitates were resolvedfully by tapping and added with 0.5 ml of 1:1.4 PEG solution [5 g ofPEG1000 (Wako Pure Chemical Industries, Ltd.), and 1 ml of DMSO (SIGMA)were dissolved in 6 ml of DMEM], and fully stirred for about 1 min and30 sec. After that, 10 ml of DMEM was slowly added, centrifuged for 10min at 1250 rpm, and suspended in 30 ml of ES culture medium.Thereafter, the cells were dispensed into three petri dishes with adiameter of 100 mm (Corning Incorporated) to which feeder cells havebeen previously added and then cultured. 24 hours later, the culturemedium was exchanged with culture medium containing 300 μg/ml G418 andthen subjected to selection culture for about 1 week. As a result, total13 colonies were isolated, amplified, and subjected to the followinganalysis. One clone from CHO (hChr21q22.12-MAC, hChr2′-hChr21q22.12) 1and one clone from CHO (hChr21q22.12-MAC, hChr2′-hChr21q22.12) 12 werepositive in PCR using the primers described above for detecting thehChr21q22.12-MAC region only. In addition, for two clones among theabove, FISH analysis (Tomizuka et al., Nature Genet. 16: 133, 1997) wascarried out by using human Cot-1 DNA and mouse minor satellite DNA. As aresult, the clones that were specifically detected with the probes andhad normal mouse nuclear type were found to be one clone (FIG. 62). Fromthe above, it was concluded that one clone of TT2F cells retaininghChr21q22.12-MAC was obtained.

[E] Stability of hChr21q22.12-MAC in Mouse ES Cell

Under non-selection culture of 0 to 50 PDL for the mouse ES clonesobtained from the above (for example, TT2F (hChr21q22.12-MAC)₈, obtainedfrom the above [D]), the rate of cells retaining hChr21q22.12-MAC afterlong-term culture was measured by FISH analysis. As a result, theretention rate of 95% or more was obtained even for 50 PDL (FIG. 63).

[F] Preparation of Chimeric Mouse Retaining hChr21q22.12-MAC

By using the ES cell clones retaining hChr21q22.12-MAC obtained from theabove [D], a chimeric mouse was prepared according to the method ofTomizuka et al. (Nature Genet. 16: 133, 1997). As a host cell,eight-cell stage embryos obtained by sexual crossbreeding of MCH (ICR)(white, purchased from CLEA Japan, Inc.) were used. Injected embryo wastransplanted into a foster mother, and coat color of the new-born mousewas examined to see whether or not it was a chimera. As the result that80 embryos injected with ES clone retaining hChr21q22.12-MAC (forexample, TT2F (hChr21q22.12-MAC)₈, obtained from the above [D]) weretransplanted into foster mothers, 43 chimeric mice (in which dark browncolor area was observed in coat color) were born. Among them, threeanimals were individuals having a chimeric rate of about 100% from whichalmost no white color area was observed. In other words, it was shownthat ES cell line (TT2F) retaining the mouse artificial chromosomevector hChr21q22.12-MAC retained a chimera forming ability, that is, anability of differentiating into normal tissue of a mouse individual.

[G] As described in Example 8, mouse lineage-based TC (hChr21q22.12-MAC)in which hChr21q22.12-MAC has been transmitted to a progeny can beprepared from the chimeric mouse retaining the mouse artificialchromosome vector hChr21q22.12-MAC. Further, by using the TC(hChr21q22.12-MAC) mouse line, stability of hChr21q22.12-MAC in somaticcells can be examined. Further, the hChr21q22.12-MAC line can be used asa model mouse for Down's syndrome, and it can be advantageously used forelucidating the mechanism for onset of Down's syndrome or developing atherapeutic agent for alleviating the symptom. Still further, bycomparing the phenotypes between TC (hChr21q-MAC) mouse line and TC(hChr21q22.12-MAC) mouse line, Down's syndrome critical gene region canbe identified.

Example 13 Stability of the Mouse Artificial Chromosome Vector MAC2

[A] Stability of the Mouse Artificial Chromosome Vector MAC2 in CHO Cell

Under non-selection culture of 0 to 25 PDL for the CHO clones obtainedfrom the above (for example, CHO(HPRT⁻; MAC2)-13 and -18, obtained fromExample 4 above), the rate of cells retaining MAC2 after long-termculture was measured by FISH analysis. As a result, the retention rateof 90% or more was obtained even for 25 PDL. In contrast, in the CHOcells retaining HAC vector (21HAC2) carrying GFP derived from chromosome21 described by Kazuki et al. (Gene Therapy: PMID: 21085194, 2010), theretention rate was 70% or less for 25 PDL. The representative resultsare given in FIG. 64.

Example 14 Construction of the Mouse Artificial Chromosome VectorFVIII-MAC

As an example of gene encoding a useful protein, Factor VIII (FVIII)gene, which is a causative gene of hemophilia A, is inserted into themouse artificial chromosome vector MAC2 by using Cre/loxP system, andexpression and long-term stability of functional protein are examined.

[A] Insertion of gene encoding certain useful protein (for example,FVIII) into the mouse artificial chromosome vector MAC2 by usingCre/loxP system in CHO cell containing the mouse artificial chromosomevector MAC2 vector.

It is examined whether or not loxP is operated and cyclic DNA can besite-specifically inserted into the mouse artificial chromosome vectorMAC2 obtained by inserting 5′ HPRT-loxP-PGKhyg type loxP sequence, as aDNA insertion sequence, into the mouse artificial chromosome vector MAC.

[A. 1] Preparation of Vector Inserted with FVIII

The promoter and poly A region of pCAGGS (provided by Dr. Okabe at OsakaUniversity) were cleaved at SalI and PstI sites, and cloned into SalIand PstI sites of pB3 obtained by modifying the multicloning site ofpBluescript KS (−) (Stratagene) (pB-CAG). B domain depleted FVIII cDNAin pKF17K plasmid (provided by Professor Sakata at Jichi MedicalUniversity) was cleaved at XhoI and SalI sites, and cloned into EcoRIsite located between the promoter and poly A of pB-CAG (pB-CAGF8).CAG-F8-pA region in pB-CAGF8 was isolated at SalI and AvrII sites, andcloned into SalI and AvrII sites so that it could be inserted into twoHS4 insulator sequences on pB3ins2 (pB3-F8ins2). Next, pPAC4 (Children'sHospital Oakland Research Institute (CHORI), BAC/PAC Resources) wasintroduced into the vector incorporated with 3′ HPRT-loxP sequence.FVIII expression cassette (HS4-CAG-F8-pA-HS4) as AscI and FseI region onpB3-F8ins2 was cloned into AscI and FseI sites of pPAC4 to give a singlecopy of FVIII insertion construct of HPRT re-construction system (vectorname: pPAC4 F8ins2 H3-9 (single copy FVIII-PAC)) (FIG. 65). By utilizingthe characteristics of compatible cohesive end of AvrII site and NheIsite, first cassette region from AscI to AvrII1 of a single copy ofFVIII-PAC was re-cloned into the region from AscI to NheI sites of thesame vector to obtain two copies of FVIII-PAC containing two expressioncassettes. Similarly, the insertion cassette was re-cloned into AscI andAvrII and the vector side was re-cloned into AscI and NheI sites toprepare 2, 4, 8, or 16 copies of PAC vector having FVIII expressioncassette (FIG. 66).

[A. 2] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection. With regard to thecells in 6 wells with 90% confluency, 1 μg of Cre and 10 μg of a singlecopy of FVIII-PAC vector were introduced into CHO(HPRT⁻; MAC2)-13described above and CHO(HPRT⁻; 21HAC2) described by Kazuki et al. (GeneTherapy: PMID: 21085194, 2010), according to the commercially availableprotocol (Invitrogen) (clone name: CHO (FVIIIx1-MAC) and CHO(FVIIIx1-HAC)). Further, 1 μg of Cre and 10 μg of 16 copies of FVIII-PACvector were introduced into CHO(HPRT⁻; MAC2)-13 described aboveaccording to the commercially available protocol (Invitrogen) (clonename: CHO (FVIIIx16-MAC)). After culture for 2 weeks under HAT selectionculture, resistant colony was generated, and total 83 colonies obtainedby four introductions, that is, 46 clones for CHO (FVIIIx1-MAC), 18clones for CHO (FVIIIx16-MAC), and 19 clones for CHO (FVIIIx1-HAC), wereisolated, amplified, and subjected to the following analysis.

[A. 3] Selection of Drug Resistant Clone

[A. 3. 1] PCR Analysis

In order to select a recombinant by using as a template genomic DNA ofHAT resistance cell line, PCR was carried out by using the followingprimers and it was confirmed whether or not site specific insertion ofFVIII gene has occurred. The primer sequences are given below.

TRANS L1 (described above) TRANS R1 (described above) FVIII F:(SEQ ID NO: 109) 5′-ATACAACGCTTTCTCCCCAA-3′ FVIII R: (SEQ ID NO: 110)5′-TCTTGAACTGAGGGACACTG-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ex Taq (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 10min, 35 cycles of 94° C. for 30 sec, 60° C. for 30 sec, and 72° C. for 1min were carried out. As a result of PCR, four clones derived from CHO(FVIIIx1-MAC), 17 clones derived from CHO (FVIIIx1-HAC), and threeclones derived from CHO (FVIIIx16-MAC) were found to be positive, andthe following analysis was performed by using those 24 clones.

[A. 3. 2] Mono-Color FISH Analysis

Based on the results above, for seven clones, mono-color FISH analysiswas carried out according to Matsubara et al. (FISH test protocol,Shujunsha Co., Ltd., 1994). FISH analysis was carried out by using mousecot-1 DNA or human cot-1 DNA as a probe. As a result, it was found thatone clone derived from CHO (FVIIIx1-MAC) retained FVIII-MAC at a rate of90% or more, two clones derived from CHO (FVIIIx1-HAC) retainedFVIII-HAC at a rate of 90% or more, and one clone derived from CHO(FVIIIx16-MAC) retained FVIII-HAC at a rate of 90% or more.

[B] Gene Expression Analysis of FVIII Gene in CHO Cell

By using CHO (FVIIIx1-MAC) 1-3 in which FVIIIx1-MAC was retained, CHO(FVIIIx1-HAC) 1-2 in which FVIIIx1-HAC was retained, and CHO(FVIIIx16-MAC) 16-1, 16-2, 16-3 in which FVIIIx16-MAC was retained,expression of FVIII mRNA was examined. Specifically, as described above,RNA was extracted, cDNA was synthesized by using the RNA as a template,and PCR was carried out by using the following primers (describedabove). Temperature and cycle conditions were as follows: after heatdenaturation at 94° C. for 10 min, 25 cycles of 94° C. for 30 sec, 60°C. for 30 sec, and 72° C. for 1 min were carried out.

FVIII F: (described above)

FVIII R: (described above)

GAPDH F: (described above)

GAPDH R: (described above)

As a result, it was found that FVIII mRNA was expressed at the samelevel in CHO (FVIIIx1-MAC) and CHO (FVIIIx1-HAC) while it was expressedmore in CHO (FVIIIx16-MAC) compared to the CHO (FVIIIx1-MAC) and CHO(FVIIIx1-HAC).

[C] Gene Function Analysis of FVIII Gene in CHO Cell

To examine whether or not the FVIII protein expression was functional inthe CHO (FVIIIx1-MAC) 1-3 and CHO (FVIIIx1-HAC) 1-2 from whichexpression of FVIII mRNA was confirmed, clotting assay (COSMO BIO CO.,LTD.) was performed according to the protocol attached thereto. Cellswere cultured under non-selection culture of 0 to 25 PDL, and culturedto a 6-well dish. At 100% confluency, the medium was exchanged with thefresh medium. 24 hours later, the culture supernatant was recovered andthe activation degree of FVIII was measured based upon the FVIIIactivity. As a result, for 0 PDL, almost no difference was found inactivity between CHO (FVIIIx1-MAC) 1-3 and CHO (FVIIIx1-HAC) 1-2. Incontrast, the activity was increased as much as 1.8 times in CHO(FVIIIx1-MAC) 1-3 even for 25 PDL, but it was decreased by 6 times inCHO (FVIIIx1-HAC) 1-2 for 25 PDL (FIG. 67). Further, compared to CHO(FVIIIx1-MAC) 1-3, the activity of CHO (FVIIIx16-MAC) 16-1, 16-2, 16-3was increased as much as about 10 times, from which it was confirmedthat the activity increased in copy number dependent manner (FIG. 68).

From the above experiments, it was confirmed that, by carrying FVIIIgene on mouse artificial chromosome MAC2 vector, functional expressionof FVIII gene was observed and functional expression was more stablyobserved for a longer period of time in CHO retaining FVIII-MAC comparedto CHO retaining FVIII-HAC. In addition, by using PAC vector, a DNA ofnot more than 200 kb which encodes a useful protein could be inserted.

Example 15 Construction of the Mouse Artificial Chromosome Vector MI-MACAllowing Multiple-Gene Introduction

As an example for describing the mouse artificial chromosome vector MAC2carrying multiple genes, multi-integrase platform having fiverecognition sites for site specific recombinase (ΦC31 attP, R4 attP,TP901-1 attP, Bxb1 attP, FRT) was inserted by using Cre/loxP system toexamine the introduction and expression of multiple genes.

[A. 1] Preparation of Multi-Integrase Platform Cassette

A cassette having multi-integrase platform for introducing multiplegenes to a mouse artificial chromosome vector was prepared as follows byusing Multisite-Gateway kit (Invitrogen). First, by using PGK-hyg(Clontech) as a template, ΦC31 attP site, R4 attP site, TP901-1 attPsite, Bxb1 attP site, and FRT site, which are the sites for geneintroduction, were added to PGK promoter sequence by first PCR (eachprimer pair F1-R1 given below). For PCR, GeneAmp 9600 manufactured byPerkinElmer, Inc. was used as a thermal cycler and KOD plus (Toyobo) wasused as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) usedwere those included in the product and they were used under theconditions described by manufacturer's instruction. Temperature andcycle conditions were as follows: after heat denaturation at 94° C. for2 min, 20 cycles of 94° C. for 15 sec, 68° C. for 30 sec, and 72° C. for90 sec were carried out. The PCR product was treated with proteinase K(Gibco) and purified by CHROMASPIN-TE400 (Clontech).

B1-FRT-PGK-ΦC31 attP-B5r F1: (SEQ ID NO: 111)5′-GAAGTTCCTATACTTTCTAGAGAATAGGAACTTCATTCTACCGGG TAGGGGAGGCGCTTTTCCC-3′B1-FRT-PGK-ΦC31 attP-B5r R1: (SEQ ID NO: 112)5′-CAACTGAGAGAACTCAAAGGTTACCCCAGTTGGGGCACTACGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3′ B5-PGK-R4 attP-B4 F1: (SEQ ID NO: 113)5′-GGGGACAACTTTGTATACAAAAGTTGATATTCTACCGGGTAGGGGA GGCGCTTTTCCC-3′B5-PGK-R4 attP-B4 R1: (SEQ ID NO: 114)5′-CACAAGCAGTACCACTGCTTCAAGTGGTATCGCTTTGGGGAACATGCGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3′ B4r-PGK-TP901 attP-B3r F1:(SEQ ID NO: 115) 5′-GGGGACAACTTTTCTATACAAAGTTGATATTCTACCGGGTAGGGGAGGCGCTTTTCCC-3′ B4r-PGK-TP901 attP-B3r R1: (SEQ ID NO: 116)5′-CTTAATTGAAATAAACGAAATAAAAACTCGCAATTAAGCGAGTTGGAAGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3′ B3-PGK-Bxb1 attP-B2 F1:(SEQ ID NO: 117) 5′-GGGGACAACTTTGTATAATAAAGTTGGTATTCTACCGGGTAGGGGAGGCGCTTTTCCC-3′ B3-PGK-Bxb1 attP-B2 R1: (SEQ ID NO: 118)5′-AGACCGCGGTGGTTGACCAGACAAACCACGAAGACACAGGTCATCACGGCCATAGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3′

By using the first PCR fragment thus obtained as a template, second PCR(each primer pair F1-R2, and F2-R2 for ΦC31 only) for adding gatewayattB sequence which is required for Multisite-Gateway BP reaction wasperformed. The PCR conditions and the like were the same as above exceptthat the cycle number was changed to 25 cycles. The primer F1 sequenceis the same as those described above.

B1-FRT-PGK-ΦC31 attP-B5r F2: (SEQ ID NO: 119)5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGAAGTTCCTAT ACTTTCTAGAGAATAGGAA-3′B1-FRT-PGK-ΦC31 attP-B5r R2: (SEQ ID NO: 120)5′-GGGGACAACTTTTGTATACAAAGTTGTGACCCTACGCCCCCAACTGAGAGAACTCAAAGGTTACCCCAGT-3′ B5-PGK-R4 attP-B4 R2: (SEQ ID NO: 121)5′-GGGGACAACTTTGTATAGAAAAGTTGGGTGCACCCGCAGAGTGTACCCACAAGCAGTACCACTGCTTCAAGTGGTAT-3′ B4r-PGK-TP901 attP-B3r R2:(SEQ ID NO: 122) 5′-GGGGACAACTTTATTATACAAAGTTGTTAAAAGGAGTTTTTTAGTTACCTTAATTGAAATAAACGAAATAAAAACTCG-3′ B3-PGK-Bxb1 attP-B2 R2:(SEQ ID NO: 123) 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTATGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCACG-3′

The PCR fragments and the donor vector (Invitrogen: pDONR221P1-P5r,pDONR221 P5-P4, pDONR221P4r-P3r, pDONR221P3-P2) having a correspondinggateway attP sequence were mixed with each other, and based on in vitrorecombination reaction using BP clonase (BP reaction), entry vectors(pENTR L1-FRT-PGK-ΦC31-R5, pENTR L5-PGK-R4-L4, pENTR R4-PGK-TP901-1-R3,pENTR L3-PGK-Bxb1-L2) were prepared (FIG. 69). BP reaction was performedaccording to the conditions as recommended.

Next, the plasmid incorporated with 3′ HPRT-loxP sequence, which isrequired for inserting the above gene insertion site into a mouseartificial chromosome vector, was prepared by the following procedure.The X3.1 was used as a template and amplification was carried out byusing the following primers. The PCR conditions are the same as theconditions described above (cycle number: 25).

PGK2362: (SEQ ID NO: 124) 5′-TGATTGTTCAGGAGGAGGAAGCCGGTGGCG-3′ loxP4548:(SEQ ID NO: 125) 5′-AGAGCCTTCAACCCAGTCAGCTCCTTCGAA-3′

The PCR fragment and DEST cassette (Invitrogen: R1-ccdB-Cm-R2) weresubjected to blunt ligation to yield pDEST. Next, the pDEST and theentry vector (pENTR L1-FRT-PGK-ΦC31-R5, pENTR L5-PGK-R4-L4, pENTRR4-PGK-TP901-1-R3, pENTR L3-PGK-Bxb1-L2) prepared above were mixed witheach other, and based on in vitro recombination reaction using LRclonase (LR reaction), the multi-integrase platform cassette wasprepared (FIG. 70). The LR reaction was performed according to theconditions as recommended.

[A. 2] Carrying Multi-Integrase Platform Cassette on the MouseArtificial Chromosome Vector

By Cre-loxP recombination into the above-described CHO(HPRT⁻; MAC2) orbelow-described CHO(HPRT⁻; MAC4) and attaining the HAT resistant clone,the multi-integrase platform cassette can be inserted into the mouseartificial chromosome vector MAC2 or MAC4 (referred to as MI-MAC) (FIG.71).

[A. 3] Preparation of Cassette for Gene Introduction

A cassette vector for introducing an exogenous gene to multi-integraseplatform was prepared as follows. First, the promoterlessneomycin-resistant gene required for drug selection was amplified byusing the following primers and pIRES Neo2 (Clontech) as a template. ThePCR conditions are the same as the conditions described above (cyclenumber: 25).

NeoF: (SEQ ID NO: 126) 5′-AAAGATATCAACTCGAGATGGGATCGGCCATTGAACAAGATGGATTG-3′ NeoR: (SEQ ID NO: 127)5′-TTTGCTAGCCCCCAGCTGGTTCTTTCCGCCTCAGAAGCC-3′

After that, the PCR fragment was blunt-cloned into SLR test (Toyobo)which has been cleaved with the restriction enzyme EcoRV and at SmaIsite to prepare pNeo. Then, the recombination sequence (ΦC31 attB, R4attB, TP901-1 attB, Bxb1 attB, FRT) corresponding to each attP site orFRT site was prepared by de novo synthesis (ΦC31, Bxb1, FRT: IntegratedDNA technologies Inc., R4, TP901-1: Invitrogen). pNeo was cleaved withthe restriction enzyme SalI, and the DNA fragment containing ΦC31 attBor R4 attB was cut from the vector synthesized above by using therestriction enzyme SalI before ligation (pNeo-ΦC31 attB, pNeo-R4 attB).Similarly, pNeo was cleaved with the restriction enzyme ClaI, the DNAfragment containing TP901-1 attB or FRT was cut out from the vectorsynthesized above by using the restriction enzyme ClaI and ligatedthereto to prepare pNeo-TP901-1 attB or pNeo-FRT, or pNeo was cleavedwith the restriction enzyme NheI, and the DNA fragment containing Bxb1attB was cut from the vector synthesized above by using the restrictionenzyme NheI and ligated thereto to prepare pNeo-Bxb1 attB. These vectorsallow insertions of any exogenous gene into BamHI site, and it is acassette vector which can be carried on a mouse artificial chromosomehaving multi-integrase platform (FIG. 72).

[A. 4] Preparation of Vector for Expression of Site Specific Recombinase

Site specific recombinases (ΦC31 integrase, R4 integrase, TP901-1integrase, Bxb1 integrase) (GenBank accession numbers: 11C31, CAA07153;R4, BAA07372; TP901-1, CAA59475; Bxb1, AAG59740), each of which cancause recombination between corresponding attB and attP, were preparedby de novo synthesis ΦC31: Codon device, others: Invitrogen). Theseintegrases were synthesized according to codon usage optimization formammals to have high expression in mammalian cells. From the vectorsynthesized, the DNA fragment containing ΦC31 integrase was cut out byusing restriction enzymes KpnI-XbaI and ligated to pVAX1 (Invitrogen)prepared by restriction with the restriction enzymes KpnI-XbaI toprepare pCMV-ΦC31, or the DNA fragment containing R4 integrase orTP901-1 integrase, Bxb1 integrase was cut out by using restrictionenzymes NheI-XhoI and ligated to pVAX1 (Invitrogen) prepared byrestriction with the restriction enzymes NheI-XhoI to prepare pCMV-R4,pCMV-TP901-1, pCMV-Bxb1 (FIG. 72).

[A. 5] Gene Transfer to MI-MAC Vector

By introducing the vector for expressing various site specificrecombinases described above instead of Cre expression vector andintroducing cassette for gene introduction instead of FVIII insertionvector, multiple (1 to 5) genes can be inserted to the mouse artificialchromosome vector MI-MAC vector. Further, as it is also possible tocarry multiple multi-integrase platform cassettes on mouse artificialchromosome MAC vector, gene can be inserted without any limitation (FIG.72).

Example 16 Construction of the Mouse Artificial Chromosome VectorPXR-MAC

Human PXR as a nuclear receptor is inserted into the mouse artificialchromosome vector MAC3 by using Cre/loxP system to construct PXR-MAC.

[A. 1] Preparation of Human PXR Insertion Vector

As a basic BAC vector for inserting human PXR gene and loxP sequence,RP11-169N13 (CHORI) containing full length human PXR gene was used.According to the method by Yamada et al. (J Hum Genet. 2008; 53 (5):447-53), Amp-5′ HPRT-loxP sequence for inserting the mouse artificialchromosome vector MAC3 was inserted into kanamycin resistant gene regionof BAC vector based on homologous recombination (vector name: PXR-loxP).

Site specific DNA insertion obtained by insertion of human PXS based onHPRT reconstruction system using Cre/loxP system is given in FIG. 73.

[A. 2] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection. To cells in 6 wellswith 90% confluency, 1 μg of Cre and 2 μg of PXR-loxP vector wereintroduced according to the commercially available protocol(Invitrogen). After culture for 2 weeks under HAT selection culture,resistant colony was generated, and total 11 colonies obtained by twointroductions were isolated, amplified, and subjected to the followinganalysis (clone name: CHO (PXR-MAC)).

[A. 3] Selection of Drug Resistant Clone

[A. 3. 1] PCR Analysis

In order to select a recombinant by using as a template genomic DNA ofHAT resistance cell line, PCR was carried out by using the followingprimers and it was confirmed whether or not site specific insertion ofPXR gene has occurred. The primer sequences are given below.

TRANS L1 (described above) TRANS R1 (described above) hPXR1L:(SEQ ID NO: 128) 5′-aaacagcaaggcaagcatcca-3′ hPXR1R: (SEQ ID NO: 129)5′-tgctttaatccagccctggtg-3′ hPXR2L: (SEQ ID NO: 130)5′-tgtttgctcaatcgtggtctcc-3′ hPXR2R: (SEQ ID NO: 131)5′-acaaaagccgaatgtggtgga-3′ hPXR3L: (SEQ ID NO: 132)5′-ccaagaggcccagaagcaaa-3′ hPXR3R: (SEQ ID NO: 133)5′-tccccacatacacggcagatt-3′ hPXR4L: (SEQ ID NO: 134)5′-acactgccaagagccgacaat-3′ hPXR4R: (SEQ ID NO: 135)5′-gcaaccttgcctctctgatggt-3′ hPXR5L: (SEQ ID NO: 136)5′-tcaaggtgtggaagggaccaa-3′ hPXR5R: (SEQ ID NO: 137)5′-acaaagcagctcggaagagga-3′ hPXR6L: (SEQ ID NO: 138)5′-gtttgttcctggggctggaat-3′ hPXR6R: (SEQ ID NO: 139)5′-caaggcaggcactttcataccc-3′ kj neo (described above)m11 6R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 10min, 35 cycles of 94° C. for 30 sec, 60° C. for 30 sec, and 72° C. for30 sec were carried out. As a result of PCR, six clones out of the 11clones were found to be positive for all primer sets, and the followinganalysis was performed by using those six clones.

[A. 3. 3] Two-Color FISH Analysis

With six clones selected from the above result, two-color FISH analysiswas carried out according to Matsubara et al. (FISH test protocol,Shujunsha Co., Ltd., 1994). FISH analysis was carried out by using mousecot-1 DNA and DNA (RP11-169N13) (CHORI) derived from human PXR-BAC asprobes. As a result, it was found that, in five clones out of the sixclones, PXR-MAC was retained at a rate of 60% or more and the signalderived from PXR-BAC was generated. Further, since no signal wasdetected from MAC3 before site specific insertion of PXR-BAC as anegative control, it was confirmed that human PXR gene wassite-specifically inserted (FIG. 74).

From the experiments above, it was possible to confirm that, by carryingthe human PXR gene on mouse artificial chromosome MAC3, CHO cellsretaining the mouse artificial chromosome vector PXR-MAC was obtained.

[B] Transfer of the Mouse Artificial Chromosome Vector PXR-MAC from CHOCell Containing the Mouse Artificial Chromosome Vector PXR-MAC to MouseES Cell

To prepare a chimeric mouse retaining PXR-MAC, introduction was carriedout from CHO cells retaining PXR-MAC obtained from the above [A] tomouse ES cells (wild type TT2F) by microcell fusion. According to themethod by Tomizuka et al. (Nature Genet. 16: 133, 1997), microcells werepurified from approximately 10⁸ cells of CHO retaining PXR-MAC(CHO(PXR-MAC) 7, 9, 10, or the like) and suspended in 5 ml of DMEM.Approximately 10⁷ mouse ES cells of TT2F were detached by trypsintreatment, washed three times with DMEM, suspended in 5 ml of DMEM, andadded to the microcells obtained by centrifugation. After centrifugationfor 10 min at 1250 rpm, the supernatant was completely removed. Theprecipitates were resolved fully by tapping and added with 0.5 ml of1:1.4 PEG solution [5 g of PEG1000 (Wako Pure Chemical Industries,Ltd.), 1 ml DMSO (SIGMA) dissolved in 6 ml of DMEM], and fully stirredfor about 1 min and 30 sec. After that, 10 ml of DMEM was slowly added,centrifuged for 10 min at 1250 rpm, and suspended in 30 ml of ES culturemedium. Thereafter, the cells were dispensed into three petri disheswith a diameter of 100 mm (Corning Incorporated) onto which feeder cellshave been previously plated and then cultured. 24 hours later, theculture medium was exchanged with culture medium containing 300 μg/mlG418 and then subjected to selection culture for about 1 week. As aresult, total 34 colonies were isolated, amplified, and subjected to thefollowing analysis. Two clones from CHO (PXR-MAC) 7, two clones from CHO(PXR-MAC) 9, and 12 clones from CHO (PXR-MAC) 10 were determined to bepositive by PCR using the primers described before for detecting thePXR-MAC region only. In addition, with the 16 clones, FISH analysis(Tomizuka et al., Nature Genet. 16: 133, 1997) was carried out by usingDNA (RP11-169N13) (CHORI) derived from human PXR-BAC. As a result, theclones that were specifically detected with the probe were found to befour clones out of the 16 clones. From the above, it was concluded thatfour clones of TT2F cells retaining PXR-MAC are obtained (FIG. 75).

[C] As described in Example 8, by preparing a chimeric mouse using themouse ES ells retaining the mouse artificial chromosome vector PXR-MAC,mouse line-based TC (PXR-MAC) in which PXR-MAC is transferred to aprogeny can be prepared. Further, by using the TC (PXR-MAC) mouse line,stability of PXR-MAC in somatic cells can be examined. Further, the TC(PXR-MAC) mouse line allows reproduction of drug-induced CYP geneexpression in human. Still further, by crossbreeding with theTC(CYP3A-MAC) mouse line, TC(CYP3A-MAC/PXR-MAC) can be prepared, and itmay be also used as a model mouse for in vivo test that is used fortesting a pharmacological effect and toxicity for development of apharmaceutical product.

Example 17 Construction of the Mouse Artificial Chromosome Vector MAC4

The mouse artificial chromosome vector MAC4 is constructed in whichGFP-5′ HPRT-loxP-PGKhyg type loxP sequence as a DNA insertion sequenceis inserted into the mouse artificial chromosome MAC. The 5′HPRT-loxP-PGKhyg type loxP sequence is inserted into HAC vector carryingGFP (21HAC2) derived from chromosome 21 described by Kazuki et al. (GeneTherapy: PMID: 21085194, 2010), and expression of HAC and MAC genes canbe compared to each other in the same vector. Further, the vector forgene introduction that is used for insertion into 21 HAC2 may be used asit is without undergoing a step for preparing a vector.

[A] Insertion of GFP-5′ HPRT-loxP-hyg Type loxP Sequence into MouseArtificial Chromosome MAC

[A. 1] Preparation of GFP-5′ HPRT-loxP-hyg Type loxP Targeting Vector

As a basic plasmid for inserting loxP sequence, pMAC2 prepared above wasused. HS4-CAG-EGFP-HS4 (provided by Dr. Okabe at Osaka University andDr. Felsenfeld at NIH), which is obtained by cutting out by using NotIand SalI, is blunted, and pMAC2 was cloned after it was cleaved withXhoI and blunting (vector name: pMAC4). The targeting vector, targetsequence, and chromosome allele obtained by homologous recombination areshown in FIG. 76.

[A. 2] Transfection and Isolation of Drug Resistant Clone

As described above, targeting vector pMAC4 prepared above was linearizedwith the restriction enzyme NotI (TAKARA), and used for transfection ofclone DT40 (MAC) prepared above. After exchanging the culture medium forculture medium containing hygromycin (1.5 mg/ml), the cells weredispensed into two 96-well culture plates and then subjected toselection culture for about 2 weeks. Total 36 resistant coloniesobtained by one transfection were isolated, amplified, and subjected tothe following analysis (clone name: DT40 (MAC4)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether or not sitespecific recombination has occurred on the mouse chromosome vector MAC.The primer sequences are given below.

m11 4L: (described above) (SEQ ID NO: 140) V907-NotI-R:5′-AGATCTCGGCTAGAGGTACCCTAGAAGATC-3′ hygF (244): (described above)m11 6R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,five clones out of the 36 clones were found to be positive for allprimer sets, and therefore the following analysis was performed by usingthose 5 clones.

[A. 3. 2] Two-Color FISH Analysis

With the five clones of DT40 (MAC4) obtained from above, two-color FISHanalysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out byusing mouse cot-1 DNA and GFP-5′ HPRT-loxP-hyg cassette as probes. As aresult, it was found that no signal derived from probe is detected inthe mouse artificial chromosome vector MAC before targeting as anegative control, while the signal derived from probe is detected at arate of 65% or more in five clones of DT40 (MAC4). Thus, it was visuallyconfirmed that site specific recombination has occurred in the fiveclones (FIG. 77). From these results, it was possible to conclude thatDT40 cell clones retaining the mouse artificial chromosome vector MAC4are obtained.

[B] Introduction of MAC4 from Chicken DT40 Cell Containing the MouseArtificial Chromosome Vector MAC4 to CHO Cell

[B. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (MAC4)-B1-5, B1-74, B2-3, and B2-4 as recipient cells,microcell fusion was carried out for CHO(HPRT⁻), which is a CHO hprtdepleted cell (obtained from the Health Science Research Resources Bank,registration number: JCRB0218), in the same manner as above. Total 23resistant colonies obtained by four microcell fusions were isolated,amplified, and subjected to the following analysis (clone name:CHO(HPRT⁻; MAC4)).

[B. 2] Selection of Drug Resistant Clone

[B. 2. 1] PCR Analysis

For extracting genomic DNA from hygromycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether or not mouseartificial chromosome MAC4 can be introduced into CHO cells. The primersequences are given below.

m11 4L: (described above)

V907-NotI-R: (described above)

hygF (244): (described above)

m11 6R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,seven clones out of the 23 clones were found to be positive for allprimer sets, and the following analysis was performed by using those 7clones.

[B. 2. 2] Mono-Color FISH Analysis

With the seven clones of CHO(HPRT⁻; MAC4) obtained from the above, FISHanalysis was carried out by using mouse Cot-1 DNA as a probe accordingto the method described by Shinohara et al. (Human Molecular Genetics,10: 1163-1175, 2001). As a result, it was confirmed that MAC4 isintroduced into CHO cells at a rate of 95% or more in four clones out ofthe seven clones (FIG. 78).

From these results, it was concluded that the mouse artificialchromosome vector MAC4 can be introduced into CHO cells.

[C] As described in Example 8, in vitro stability can be examined bypreparing mouse ES cells retaining the mouse artificial chromosomevector MAC4 and using it. Further, by preparing a chimeric mouse usingthe ES cells, mouse line-based TC (MAC4) in which MAC4 is transmitted toa progeny can be prepared. Still further, by using the TC (MAC4) mouseline, stability of MAC4 in somatic cells can be examined.

Example 18 Construction of the Mouse Artificial Chromosome VectorUGT2-MAC

UGT2 cluster which is a group of human drug metabolizing enzyme genes issubjected to translocation cloning into the mouse artificial chromosomevector MAC4 by using Cre/loxP system to construct UGT2-MAC in the samemanner as in Example 3. Further, stability of UGT2-MAC in the mouse EScells is examined.

[A] Site Specific Cleavage at AC125239 on Human Chromosome 4

To delete the gene at the distal side from UGT2 gene cluster of humanchromosome 4, telomere truncation, which is site specific deletion of achromosome, is performed.

[A. 1] Preparation of Targeting Vector pTELpuro-UGT2

Targeting vector pTELpuro-UGT2 for inserting human telomere sequenceinto AC125239 region, which is located extremely close to UGT2 genelocus of human chromosome 4 and on the telomere side (i.e., locating onthe telomere side by approximately 150 Kb from UGT2 gene locus), wasprepared as follows. First, the AC125239 genome region was amplified byPCR using the following primers.

(SEQ ID NO: 141) UGT2tel4L; 5′- ttctggcaagccttgaagggacaatact-3′(SEQ ID NO: 142) UGT2tel4R; 5′- gcctattttgcctcataacccactgctc-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 20 sec and 68° C. for 8 min were carried out. PCR product wastreated with proteinase K (Gibco) and subjected to gel filtration byusing CHROMASPIN-TE400 (Clontech). After that, the product was cleavedwith the restriction enzyme PstI (NIPPON GENE CO., LTD.) and BglII(NIPPON GENE CO., LTD.) and subjected to gel filtration by usingCHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the PstIand BamHI sites of plasmid pTELpuro (Kuroiwa et al., Nature Biotech.,20: 88, 2002). Since the genome sequence of AC125239 was in direction ofcentromere→telomere, the resultant in which cloned AC125239 genomefragment was in the same direction as the human telomere sequence wastaken as desired targeting vector pTELpuro-UGT2. The size of the finalconstruct for long-arm proximal region specific cleavage was 11.9 kb.The targeting vector, target sequence, and chromosome allele obtained byhomologous recombination are shown in FIG. 79.

[A. 2] Transfection and Isolation of Drug Resistant Clone

According to the method described by Kazuki et al. BBRC 2004, chickenDT40 cells retaining human chromosome 4 were prepared from A9 (KM64-4)retaining human chromosome 4 (Kugoh et al. DNA research 1999) (clonename: DT40 (hChr4)). Next, as described above, the targeting vectorpTELpuro-UGT2 prepared above was linearized with the restriction enzymePstI (NIPPON GENE CO., LTD.), and used for transfection of the cloneDT40 (hChr4) 1 prepared above. After exchanging the culture medium forculture medium containing puromycin (0.3 ug/ml), the cells weredispensed into ten 96-well culture plates and then subjected toselection culture for about 2 weeks. Total 96 resistant coloniesobtained by four transfections were isolated, amplified, and subjectedto the following analysis (clone name: DT40 (hChr4-tel)).

A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

In order to select a recombinant by using genomic DNA of puromycinresistant cell line as a template, as a primary screening, PCR wascarried out by using the following primers that are located closer tothe telomere side than the restriction sites, and it was confirmedwhether or not site specific cleavage has occurred. The primer sequencesare given below.

(SEQ ID NO: 143) CSN1S1-1L; 5′-tttctcctctcaaggaaaacca-3′(SEQ ID NO: 144) CSN1S1-1R; 5′-gccctccatatggcaagaca-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 95° C. for 10min, 30 cycles of 95° C. for 20 sec, 55° C. for 30 sec, and 72° C. for30 sec were carried out. Next, for two clones that were not detectedwith the above primers, it was confirmed, by PCR using the followingprimers, whether or not site specific homologous recombination hasoccurred. Sequences are as follows.

UGT2tel4L; (described above) (SEQ ID NO: 145) SK23:5′-ggccgctctagaactagtggatc-3′ (SEQ ID NO: 146) UGT2A1-1L:5′-tcttctgcatcaagccacatca-3′ (SEQ ID NO: 147) UGT2A1-1R:5′-agccaatgactaccttccattg-3′ (SEQ ID NO: 148) UGT2A1-2L:5′-atcagggagccaccgtagga-3′ (SEQ ID NO: 149) UGT2A1-2R:5′-gcaggcaagttatgccgtga-3′ (SEQ ID NO: 150) UGT2A3-1L:5′-tgcgcccaaacacatggata-3′ (SEQ ID NO: 151) UGT2A3-1R:5′-tggcagaaatgtaggccatga-3′ (SEQ ID NO: 152) UGT2B4-1L:5′-aggctggaagctgggaaacc-3′ (SEQ ID NO: 153) UGT2B4-1R:5′-cctgcatgaaatggatccaaag-3′ (SEQ ID NO: 154) UGT2B7-1L:5′-ccagcaagaaagattgtgatgc-3′ (SEQ ID NO: 155) UGT2B7-1R:5′-ttctaaccatgaactgggtggt-3′ (SEQ ID NO: 156) UGT2B11-1L:5′-gggtttctgctggcctgtgt-3′ (SEQ ID NO: 157) UGT2B11-1R:5′-tctggttttccagcttcaaatg-3′ (SEQ ID NO: 158) UGT2B15-1L:5′-ggtctccttggcatgcacct-3′ (SEQ ID NO: 159) UGT2B15-1R:5′-tgcaatgcttcttttccagttg-3′ (SEQ ID NO: 160) UGT2B15-2L:5′-cagcatggagggttttaaatgg-3′ (SEQ ID NO: 161) UGT2B15-2R:5′-atgttggcgtgctgcatcc-3′ (SEQ ID NO: 162) UGT2B28-1L:5′-catttgaagctggaaaaccaga-3′ (SEQ ID NO: 163) UGT2B28-1R:5′-cctgggtggtaaatctctgaaa-3′

For PCR, LA Taq (TAKARA SHUZO CO., LTD.) was used with the aboveprimers. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 8 min were carried out.Only in two clones having site specific recombination, a band atapproximately 8 kb was detected. In DT40 and DT40 (hChr4) 1 as anegative control, no band was detected.

[A. 3. 2] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out fortwo clones out of the clones which have been confirmed to haverecombination in the above by using human cot-1 DNA and puromycin DNA asprobes. As a result, it was found that human chromosome 4 was nottranslocated to the host chromosome in any clone, and based on the factthat puromycin-derived signal was detected at the terminal of humanchromosome 4 fragment and restriction occurred on the desired site, itwas confirmed that recombination has site-specifically occurred (FIG.80).

From these results, it was concluded that, in clone DT40 (hChr4-tel) 35and 73, cleavage can be made at a region distal from AC125239 which iscloser to the telomere side than UGT2 gene cluster region.

[B] Site Specific Insertion of loxP Sequence into AC074378 of HumanChromosome 4

For translocation insertion into the mouse artificial chromosome vectorMAC4 via loxP sequence, loxP sequence is inserted into AC074378 proximalto UGT2 gene cluster of hChr4-tel in DT40 cells.

[B. 1] Preparation of Targeting Vector pUGT2loxPneo

Targeting vector pUGT2loxPneo for inserting loxP, which is a recognitionsequence for Cre recombinase, into AC074378 region, which is locatedextremely close to UGT2 gene locus of human chromosome 4 and on thecentromere side (i.e., locating on the centromere side by approximately300 Kb from UGT2 gene locus), was prepared as follows. First, theAC074378 genome region was amplified by PCR using the following primers.

(SEQ ID NO: 164) UGT2loxP3L: 5′- ggaacaatcccaatcaaaacctcagtgc-3′(SEQ ID NO: 165) UGT2loxP4R: 5′- cgaggattcaagccacatccctaactct-3′

As a basic plasmid for inserting loxP sequence, V907 (Lexicon genetics)was used. For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. wasused as a thermal cycler and LA Taq (TAKARA SHUZO CO., LTD.) was used asTaq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used werethose included in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 7 min were carried out.PCR product was treated with proteinase K (Gibco) and subjected to gelfiltration by using CHROMASPIN-TE400 (Clontech). After that, the productwas cleaved with the restriction enzymes KpnI (NIPPON GENE CO., LTD.),EcoRI (NIPPON GENE CO., LTD.), and BglII (NIPPON GENE CO., LTD.) andsubjected to gel filtration by using CHROMASPIN-TE1000 (Clontech). ThePCR fragments (2.7 kb and 2.6 kb) were cloned into the KpnI or EcoRI andBglII sites of V907 plasmid (vector name: V907-UGT2HR2). Next,FRT-pGKneo-FRT was cleaved from pNT1.1, which isloxP-FRT-pGKneo-FRT-loxP cassette (obtained from Genome InformationResearch Center, Osaka University), with EcoRI and BamHI and cloned intoBglII site of the X3.1 (vector name: X3.1-FRT-pGKneo-FRT). Thereafter,V907-UGT2HR2 was cleaved with the restriction enzyme EcoRI and the DNAfragment containing loxP was cut out from X3.1-FRT-pGKneo-FRT by usingthe restriction enzyme EcoRI, and then they were ligated to each other.The resultant product having the loxP sequence in the same direction asthe cloned AC074378 genome fragment was taken as targeting vectorpUGT2loxPneo. Size of the final construct inserted with loxP was 11.1kb. The targeting vector, target sequence, and chromosome alleleobtained by homologous recombination are shown in FIG. 81.

[B. 2] Transfection and Isolation of Drug Resistant Clone

As described above, the targeting vector pUGT2loxPneo prepared above waslinearized with the restriction enzyme NotI (TAKARA), and used fortransfection of the chicken DT40 cells retaining human chromosome 4(clone DT40 (hChr4-tel) 35. After exchanging the culture medium with aculture medium containing neomycin (1.5 mg/ml), the cells were dispensedinto three 96-well culture plates and then subjected to selectionculture for about 2 weeks. Total 12 resistant colonies obtained by twotransfections were isolated, amplified, and subjected to the followinganalysis (clone name: DT40 (hChr4-tel-loxP)).

B. 3] Selection of Homologous Recombinant

[B. 3. 1] PCR Analysis

Genomic DNA was extracted from the neomycin resistant clones by usingPuregene DNA Isolation Kit (Gentra Systems, Inc.) and identification ofthe homologous recombinant was carried out by PCR using the followingtwo sets of primer.

Identification of the homologous recombinants was carried out by PCRusing the following two sets of primer.

UGT2loxP3L (described above)

TRANS R1 (described above)

PGKr1 (described above)

UGT2loxP4R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 4 min were carried out. As a result ofscreening 12 clones, five clones were identified as a homologousrecombinant.

[B. 3. 2] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). FISH analysis was carried out forfour clones out of the clones which have been confirmed to haverecombination in the above by using human cot-1 DNA and neomycin DNA asprobes. As a result, it was found that human chromosome 4 was nottranslocated to the host chromosome in any clone, and based on the factthat neomycin-derived signal was detected near 4q13, it was confirmedthat recombination has site-specifically occurred (FIG. 82). From theseresults, it was concluded that loxP sequence as a gene introduction siteis site-specifically inserted into AC074378 of human chromosome 4.

[C] Introduction of hChr4-loxP-tel from DT40 Containing hChr4-loxP-telto CHO Cells Containing MAC4

For translocation insertion of human UGT2 gene cluster region into themouse artificial chromosome vector MAC4 via loxP sequence in CHO cells,hChr4-loxP-tel is introduced into CHO cells containing the mouseartificial chromosome vector MAC4.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (hChr4-loxP-tel) 5 and 10 as a recipient cell, microcellfusion was carried out for CHO(HPRT⁻; MAC4), which is a CHO hprtdepleted cell containing MAC4 (obtained from the Health Science ResearchResources Bank, registration number: JCRB0218), in the same manner asabove. Total 22 resistant colonies obtained by three microcell fusionswere isolated, amplified, and subjected to the following analysis (clonename: CHO(HPRT⁻; MAC4, hChr4-loxP-tel)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from neomycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether or not humanchromosome 4 fragment is introduced into CHO cells containing MAC4. Theprimer sequences are given below.

m11 4L: (described above)

V907-NotI-R: (described above)

hygF (244): (described above)

m11 6R (described above)

UGT2tel4L; (described above)

SK23 (described above)

UGT2A1-1L (described above)

UGT2A1-1R (described above)

UGT2A1-2L (described above)

UGT2A1-2R (described above)

UGT2A3-1L (described above)

UGT2A3-1R (described above)

UGT2B4-1L (described above)

UGT2B4-1R (described above)

UGT2B7-1L (described above)

UGT2B7-1R (described above)

UGT2B11-1L (described above)

UGT2B11-1R (described above)

UGT2B15-1L (described above)

UGT2B15-1R (described above)

UGT2B15-2L (described above)

UGT2B15-2R (described above)

UGT2B28-1L (described above)

UGT2B28-1R (described above)

UGT2loxP3L (described above)

TRANS R1 (described above)

PGKr1 (described above)

UGT2loxP4R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,five clones out of the 22 clones were found to be positive for allprimer sets, and the following analysis was performed by using thosefive clones.

[C. 2. 2] Two-Color FISH Analysis

For the five clones of CHO(HPRT⁻; MAC4, hChr4-loxP-tel) obtained fromthe above, FISH analysis was carried out by using mouse Cot-1 DNA andhuman Cot-1 DNA as probes according to the method described by Shinoharaet al. (Human Molecular Genetics, 10: 1163-1175, 2001). As a result, itwas confirmed that one copy or two copies of MAC1 and hChr4-loxP-telwere introduced into CHO cells with a rate of 90% or more in one clone(FIG. 83).

From these results, it was concluded that hChr4-loxP-tel could beintroduced into the CHO cells containing the mouse artificial chromosomevector MAC4.

[D] Site Specific Translocation of 2 Mb Human UGT2 Gene Cluster Region(i.e., AC074378-Human UGT2 Gene Cluster-AC125239) to MAC4 Vector inCHO(HPRT⁻; MAC4, hChr4-loxP-tel) Clone

To stably keep the human UGT2 gene cluster, which is a DNA with 2 Mbsize, in a mouse individual, translocation insertion into the mouseartificial chromosome vector MAC4 is performed (FIG. 84).

[D. 1] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection for CHO(HPRT⁻; MAC4,hChr4-loxP-tel) 8 obtained from the above. To cells in 6 wells with 90%confluency, 3 μg of Cre was introduced according to the commerciallyavailable protocol (Invitrogen). After culture for 2 weeks under HATselection culture, a resistant colony was generated and total sixcolonies obtained by two introductions were isolated, amplified, andsubjected to the following analysis (clone name: CHO (UGT2-MAC,hChr4-ΔUGT2)).

[D. 2] Selection of Drug Resistant Clone

[D. 2. 1] PCR Analysis

For extracting genomic DNA from HAT resistant cell line and using it asa template for selecting a clone with reciprocal translocation, PCR wascarried out by using the following primers and it was confirmed whetheror not reciprocal chromosomal translocation has occurred on humanchromosome 4 fragment and MAC4. The primer sequences are given below.

m11 4L: (described above)

V907-NotI-R: (described above)

hygF (244): (described above)

m11 6R (described above)

UGT2tel4L; (described above)

SK23 (described above)

UGT2A1-1L (described above)

UGT2A1-1R (described above)

UGT2A1-2L (described above)

UGT2A1-2R (described above)

UGT2A3-1L (described above)

UGT2A3-1R (described above)

UGT2B4-1L (described above)

UGT2B4-1R (described above)

UGT2B7-1L (described above)

UGT2B7-1R (described above)

UGT2B11-1L (described above)

UGT2B11-1R (described above)

UGT2B15-1L (described above)

UGT2B15-1R (described above)

UGT2B15-2L (described above)

UGT2B15-2R (described above)

UGT2B28-1L (described above)

UGT2B28-1R (described above)

UGT2loxP3L (described above)

TRANS R1 (described above)

PGKr1 (described above)

UGT2loxP4R (described above)

TRANS L1 (described above)

TRANS R1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,all six clones out of the six clones were found to be positive for allprimer sets, and the following analysis was performed by using those sixclones.

[D. 2. 2] Two-Color FISH Analysis

With the six clones of CHO (UGT2-MAC, hChr4-ΔUGT2) obtained from theabove, FISH analysis was carried out by using UGT2-BAC(RP11-643N16)(CHORI) DNA and mouse Cot-1 DNA as probes according to the methoddescribed by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175,2001). As a result, it was confirmed that the signal derived from humanUGT2 was observed on MAC4 at a rate of 50% or more in two clones out ofthe six clones (FIG. 85).

From these results, it was concluded that 2 Mb of UGT2 cluster on humanchromosome 4 fragment could be cloned into the mouse artificialchromosome vector MAC4 by reciprocal translocation.

[E] Transfer of UGT2-MAC from CHO Cell to Mouse A9 Cell

To prepare mouse ES cells retaining UGT2-MAC, transfer was carried outfrom CHO cells retaining UGT2-MAC(CHO (UGT2-MAC, hChr4-ΔUGT2) 4, 5)obtained from the above [D] to, as a mouse A9 cell, mouse A9 cellshaving high microcell forming ability by microcell fusion. Total 16resistant colonies obtained by four microcell fusions were isolated,amplified, and subjected to the following analysis (clone name: A9(UGT2-MAC)). As a result, there were five clones which were determinedto be positive by PCR using the primers described above for detectingthe UGT2-MAC region only. In addition, FISH analysis (Tomizuka et al.,Nature Genet. 16: 133, 1997) was carried out by using UGT2-BAC(RP11-643N16) (CHORI) and mouse minor satellite DNA as probes. As aresult, the presence of UGT2-MAC, which is specifically detected withthe probes, was confirmed in all five clones out of the five clones(FIG. 86). From the above, it was concluded that five clones of A9 cellsretaining UGT2-MAC were obtained.

[F] Transfer of UGT2-MAC from A9 Cell to Mouse ES Cell

To prepare a chimeric mouse retaining UGT2-MAC, introduction was carriedout from A9 cells retaining UGT2-MAC obtained from the above [E] tomouse ES cells (wild type TT2F) by microcell fusion. According to themethod of Tomizuka et al. (Nature Genet. 16: 133, 1997), microcells werepurified from approximately 10⁸ cells of A9 retaining UGT2-MAC (A9(UGT2-MAC) 13, 15, or the like) and suspended in 5 ml of DMEM.Approximately 10⁷ mouse ES cells were removed by trypsin treatment,washed three times with DMEM, suspended in 5 ml of DMEM, and added tothe microcells obtained by centrifugation. After centrifugation for 10min at 1250 rpm, the supernatant was completely removed. Theprecipitates were resolved fully by tapping and added with 0.5 ml of1:1.4 PEG solution [5 g of PEG1000 (Wako Pure Chemical Industries, Ltd.)and 1 ml of DMSO (Sigma) are dissolved in 6 ml of DMEM], and fullystirred for about 1 min and 30 sec. After that, 10 ml of DMEM was slowlyadded, centrifuged for 10 min at 1250 rpm, and suspended in 30 ml of ESculture medium. Thereafter, the cells were dispensed into three petridishes with a diameter of 100 mm (Corning Incorporated) onto whichfeeder cells have been previously plated and then cultured. 24 hourslater, the culture medium was exchanged with culture medium containing300 μg/ml G418 and then subjected to selection culture for about 1 week.As a result, total 25 colonies were isolated, amplified, and subjectedto the following analysis. Five clones from A9 (UGT2-MAC) 13 and fourclones from A9 (UGT2-MAC) 15 were positive in PCR using the primersdescribed before for detecting the UGT2-MAC region only. In addition,with nine clones among the above, FISH analysis (Tomizuka et al., NatureGenet. 16: 133, 1997) was carried out by using UGT2-BAC(RP11-643N16)(CHORI) and mouse minor satellite DNA. As a result, the clones that arespecifically detected with the probes and have normal mouse nuclear typewere found to be seven clones (FIG. 87). From the above, it wasconcluded that seven clones of TT2F cells retaining UGT2-MAC wereobtained.

[G] Stability of UGT2-MAC in Mouse ES Cells

Under non-selection culture of 0 to 50 PDL for the mouse ES clonesobtained from the above (for example, TT2F (UGT2-MAC) 9, 10, 19,obtained from the above [F]), a rate of cells retaining UGT2-MAC afterlong-term culture was measured by FISH analysis. As a result, theretention rate of 95% or more was obtained even for 50 PDL (FIG. 88).

[H] As described in Example 8, by preparing the chimeric mouse using themouse ES cells retaining the mouse artificial chromosome vectorUGT2-MAC, mouse line-based TC (UGT2-MAC) in which UGT2-MAC has beentransmitted to a progeny can be prepared. Further, by using the TC(UGT2-MAC) mouse line, stability of UGT2-MAC in somatic cells can beexamined. Still further, since human drug metabolism can be reproducedby the TC (UGT2-MAC) mouse line, it can be used as a model mouse for invivo test that is used for testing a pharmacological effect and toxicityin the phase II reaction for development of a pharmaceutical product.

Example 19 Construction of the Mouse Artificial Chromosome VectorCYP2C-MAC

Translocation cloning of CYP2C cluster, which is a human drugmetabolizing enzyme gene group into the mouse artificial chromosomevector MAC4 is performed by using Cre/loxP system to construct CYP2C-MACin the same manner as in Example 3.

[A] Site Specific Cleavage at AL157834 on Human Chromosome 10

To delete genes existing on the distal side from CYP2C gene cluster ofhuman chromosome 10, telomere truncation, which is site specificdeletion of a chromosome, is performed.

[A. 1] Preparation of Targeting Vector pTELpuro-CYP2C

Targeting vector pTELpuro-CYP2C for inserting human telomere sequence toAL157834 region, which is located extremely close to CYP2C gene locus ofhuman chromosome 10 and on the telomere side (i.e., locating on thetelomere side by approximately 150 Kb from CYP2C gene locus), wasprepared as follows. First, the AL157834 genome region was amplified byPCR using the following primers.

(SEQ ID NO: 166) 2Ctel2L; 5′- GCTATGAGACACAGGGCAGCTGAAAGTC-3′(SEQ ID NO: 167) 2Ctel2R; 5′- TTGTGAACCACCATGCCTAGCTGAAAGT-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 20 sec and 68° C. for 8 min were carried out. PCR product wastreated with proteinase K (Gibco) and subjected to gel filtration byusing CHROMASPIN-TE400 (Clontech). After that, the product was cleavedwith the restriction enzymes BamHI (NIPPON GENE CO., LTD.) and BglII(NIPPON GENE CO., LTD.) and subjected to gel filtration by usingCHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the BamHIsite of plasmid pTELpuro (Kuroiwa et al., Nature Biotech., 20: 88,2002). Since the genome sequence of AL157834 is in the direction ofcentromere→telomere, the resultant in which cloned AL157834 genomefragment is in the same direction as the human telomere sequence wastaken as desired targeting vector pTELpuro-CYP2C. Size of the finalconstruct for long-arm proximal region specific restriction was 11.6 kb.The targeting vector, target sequence, and chromosome allele obtained byhomologous recombination are shown in FIG. 89.

[A. 2] Transfection and Isolation of Drug Resistant Clone

According to the method described by Kazuki et al. BBRC 2004, chickenDT40 cells retaining human chromosome 10 were prepared from A9 (KM32-2)and A9 (KM26-3) (Kugoh et al. DNA research 1999) retaining humanchromosome 10 (clone name: DT40 (hChr10)). Next, as described above, thetargeting vector pTELpuro-CYP2C prepared above was linearized with therestriction enzyme PstI (NIPPON GENE CO., LTD.), and used fortransfection of the clone DT40 (hChr10) 1, 42 prepared above. Afterexchanging the culture medium for culture medium containing puromycin(0.3 μg/ml), the cells were dispensed into ten 96-well culture platesand then subjected to selection culture for about 2 weeks. Total 96resistant colonies obtained by four transfections were isolated,amplified, and subjected to the following analysis (clone name: DT40(hChr10-tel)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

In order to select a recombinant by using as a template the genomic DNAof puromycin resistant cell line, as a primary screening, PCR wascarried out by using the following primers that are located closer tothe telomere side than the restriction sites, and it was confirmedwhether or not site specific cleavage has occurred. The primer sequencesare given below.

(SEQ ID NO: 168) h10yi_1F; 5′- ACGGGGCTCCTACTCTTGTC-3′ (SEQ ID NO: 169)h10yi_1R; 5′- GCTTCCACCTGCATCTCAC-3′ (SEQ ID NO: 170) h10yi_2F;5′- CAATGCCTTATGCATGTTGTG-3′ (SEQ ID NO: 171) h10yi_2R;5′- TCCACAGCATACTGCTGACC-3′ (SEQ ID NO: 172) h10yi_3F;5′- AAGGAAGGTGACCGCCTACT-3′ (SEQ ID NO: 173) h10yi_3R;5′- CATCCGAGGACATCTTTGGT-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 95° C. for 10min, 30 cycles of 95° C. for 20 sec, 55° C. for 30 sec, and 72° C. for30 sec were carried out. Next, for three clones that are not detectedwith the above primers, it was confirmed, by PCR using the followingprimers, whether or not site specific homologous recombination hasoccurred. Sequences are as follows.

2Ctel4L; (SEQ ID NO: 174) 5′-ATCTGCAGGGAAGGGATCCAGTTTCAGCTTCCTAC-3′ SK23(described above) CYP2C8-1F: (SEQ ID NO: 175) 5′-ACATGTCAAAGAGACACACA-3′CYP2C8-1R: (SEQ ID NO: 176) 5′-TAGCATATTTCCAATAATAGGA-3′ CYP2C9-1F:(SEQ ID NO: 177) 5′-AGAAGGCTTCAATGGATTCTC-3′ CYP2C9-1R: (SEQ ID NO: 178)5′-TGTCCTTAATACCTATCTGTAGG-3′ CYP2C18-1F: (SEQ ID NO: 179)5′-ACAGCTGGATCCATTGAAGG-3′ CYP2C19-1F: (SEQ ID NO: 180)5′-ACACACACTTAATTAGCATGGA-3′ CYP2C19-1R: (SEQ ID NO: 181)5′-TTGGTTAAGGATTTGCTGACA-3′

For PCR, LA Taq (TAKARA SHUZO CO., LTD.) was used with the aboveprimers. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 8 min were carried out.Only in two clones having site specific recombination, a band atapproximately 8 kb was detected. In DT40 and DT40 (hChr10) 1, 42 as anegative control, no band was detected.

[B. 3. 2] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). With three clones which have beenconfirmed to have recombination in the above, FISH analysis was carriedout by using human cot-1 DNA and puromycin DNA as probes. As a result,it was found that human chromosome 10 was not translocated to the hostchromosome in any clone, and based on the fact that puromycin-derivedsignal was detected at the terminal of human chromosome 10 fragment andrestrictions occurred at desired sites, it was confirmed thatrecombination has site-specifically occurred (FIG. 90).

From these results, it was concluded that, in clone DT40 (hChr10-tel) 5,98, and 101, cleavage can be made at a region distal from AL157834 whichis closer to the telomere side than CYP2C gene cluster region.

[B] Site Specific Insertion of loxP Sequence to AL138759 of HumanChromosome 10

For translocation insertion into the mouse artificial chromosome vectorMAC4 via loxP sequence, loxP sequence is inserted into AL138759 proximalto CYP2C gene cluster of hChr10-tel in DT40 cells.

[B. 1] Preparation of Targeting Vector pCYP2CloxPneo

Targeting vector pCYP2CloxPneo for inserting loxP as a recognitionsequence for Cre recombinase into AL138759 region, which is locatedextremely close to CYP2C gene locus of human chromosome 10 and on thecentromere side (i.e., locating on the centromere side by approximately300 Kb from CYP2C gene locus), was prepared as follows. First, theAL138759 genome region was amplified by PCR using the following primers.

hloxP-SacII-EcoRI-F: (SEQ ID NO: 182)5′-TCCCCGCGGATCTGCTCCATACTCTGTACC-3′ hloxP-1R: (SEQ ID NO: 183)5′-CATTCAAGGGGTTCTGGGTCTGTAAACT-3′

As a basic plasmid for inserting loxP sequence, V907 (Lexicon genetics)was used. For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. wasused as a thermal cycler and LA Taq (TAKARA SHUZO CO., LTD.) was used asTaq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used werethose included in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 7 min were carried out.PCR product was treated with proteinase K (Gibco) and subjected to gelfiltration by using CHROMASPIN-TE400 (Clontech). After that, the productwas cleaved with the restriction enzymes SacII (NIPPON GENE CO., LTD.),EcoRI (NIPPON GENE CO., LTD.), and BamHI (NIPPON GENE CO., LTD.) andsubjected to gel filtration by using CHROMASPIN-TE1000 (Clontech). ThePCR fragments (1.5 kb and 3.0 kb) were cloned into the SacII and EcoRIor EcoRI and BamHI sites of V907 plasmid (vector name: V907-CYP2CHR2).Next, V907-CYP2CHR2 was cleaved with the restriction enzyme EcoRI, andthe DNA fragment containing loxP was cut out from theX3.1-FRT-pGKneo-FRT by using the restriction enzyme EcoRI, and then theywere ligated to each other. The resultant having the loxP sequence inthe same direction as the cloned AL138759 genome fragment was taken astargeting vector pCYP2CloxPneo. Size of the final construct insertedwith loxP is 10.3 kb. The targeting vector, target sequence, andchromosome allele obtained by homologous recombination are shown in FIG.91.

[B. 2] Transfection and Isolation of Drug Resistant Clone

As described above, the targeting vector pCYP2CloxPneo prepared abovewas linearized with the restriction enzyme NotI (TAKARA), and used fortransfection of the chicken DT40 cells (clone DT40 (hChr10-tel) 1-98retaining human chromosome 10. After exchanging the culture medium forculture medium containing neomycin (1.5 mg/ml), the cells were dispensedinto three 96-well culture plates and then subjected to selectionculture for about 2 weeks. Total 15 resistant colonies obtained by twotransfections were isolated, amplified, and subjected to the followinganalysis (clone name: DT40 (hChr10-tel-loxP)).

[B. 3] Selection of Homologous Recombinant

[B. 3. 1] PCR Analysis

Genomic DNA was extracted from the neomycin resistant clone by usingPuregene DNA Isolation Kit (Gentra Systems, Inc.) and identification ofthe homologous recombinants was carried out by PCR using the followingtwo sets of primer.

Identification of the homologous recombinants was carried out by PCRusing the following two sets of primer.

hloxP-SacII-EcoRI-F (described above)

TRANS R1 (described above)

PGKr 1 (described above)

hloxP-1R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 4 min were carried out. As a result ofscreening 15 clones, one clone was identified as a homologousrecombinant.

[B. 3. 2] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). With one clone which have beenconfirmed to have recombination in the above, FISH analysis was carriedout by using human cot-1 DNA and neomycin as probes. As a result, it wasfound that human chromosome 10 was not translocated to the hostchromosome in any clone, and based on the fact that neomycin-derivedsignal was detected near 10q24, it was confirmed that recombination hassite-specifically occurred. From these results, it was concluded thatloxP sequence as a gene introduction site was inserted site-specificallyinto AL138759 of human chromosome 10.

[C] Introduction of hChr10-loxP-tel from DT40 Containing hChr10-loxP-telto CHO Cell Containing MAC4

For translocation insertion of human CYP2C gene cluster region into themouse artificial chromosome vector MAC4 via loxP sequence in CHO cells,hChr10-loxP-tel is introduced into CHO cells containing the mouseartificial chromosome vector MAC4.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (hChr10-loxP-tel) 7 as a recipient cell, microcell fusionwas carried out for CHO(HPRT⁻; MAC4), which is CHO hprt depleted cellscontaining MAC4 (obtained from the Health Science Research ResourcesBank, registration number: JCRB0218), in the same manner as above. Totaleight resistant colonies obtained by three microcell fusions wereisolated, amplified, and subjected to the following analysis (clonename: CHO(HPRT⁻; MAC4, hChr10-loxP-tel)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from neomycin resistant cell line and usingit as a template for selecting a recombinant, PCR was carried out byusing the following primers and it was confirmed whether or not humanchromosome 10 fragment is introduced into CHO cells containing MAC4. Theprimer sequences are given below.

m11 4L: (described above)

V907-NotI-R: (described above)

hygF (244): (described above)

m11 6R (described above)

2Ctel4L (described above)

SK23 (described above)

CYP2C8-1F (described above)

CYP2C8-1R (described above)

CYP2C9-1F (described above)

CYP2C9-1R (described above)

CYP2C18-1F (described above)

CYP2C19-1F (described above)

CYP2C19-1R (described above)

hloxP-SacII-EcoRI-F (described above)

TRANS R1 (described above)

PGKr1 (described above)

hloxP-1R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,all eight clones out of the eight clones were found to be positive forall primer sets, and the following analysis was performed by using thoseeight clones.

[C. 2. 2] Two-color FISH analysis

With the eight clones of CHO(HPRT⁻; MAC4, hChr10-loxP-tel) obtained fromthe above, FISH analysis was carried out by using mouse Cot-1 DNA andhuman Cot-1 DNA as probes according to the method described by Shinoharaet al. (Human Molecular Genetics, 10: 1163-1175, 2001). As a result, itwas confirmed that one or two copies of MAC1 and hChr10-loxP-tel areintroduced into CHO cells with a rate of 90% or more in two clones (FIG.92).

From these results, it was concluded that hChr10-loxP-tel can beintroduced into CHO cells containing the mouse artificial chromosomevector MAC4.

[D] Site specific translocation of 380 kb human CYP2C gene clusterregion (i.e., AL138759-human CYP2C gene cluster-AL157834) to MAC4 vectorin CHO(HPRT⁻; MAC4, hChr10-loxP-tel) clone

To stably keep the human 2CYP2C gene cluster, which is a 380 kb DNA, ina mouse individual, translocation insertion into the mouse artificialchromosome vector MAC4 is performed (FIG. 93).

[D. 1] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection for CHO(HPRT⁻; MAC4,hChr10-loxP-tel) 1 and 5 obtained from the above. To cells in 6 wellswith 90% confluency, 3 μg of Cre was introduced according to thecommercially available protocol (Invitrogen). After culture for 2 weeksunder HAT selection culture, a resistant colony was generated and total11 colonies obtained by two introductions were isolated, amplified, andsubjected to the following analysis (clone name: CHO (CYP2C-MAC,hChr10-ΔCYP2C)).

[D. 2] Selection of Drug Resistant Clone

[D. 2. 1] PCR Analysis

For extracting genomic DNA from HAT resistant cell line and using it asa template for selecting a clone with reciprocal translocation, PCR wascarried out by using the following primers and it was confirmed whetheror not reciprocal chromosomal translocation has occurred on humanchromosome 10 fragment and MAC4. The primer sequences are given below.

m11 4L: (described above)

V907-NotI-R: (described above)

hygF (244): (described above)

m11 6R (described above)

2Ctel4L (described above)

SK23 (described above)

CYP2C8-1F (described above)

CYP2C8-1R (described above)

CYP2C9-1F (described above)

CYP2C9-1R (described above)

CYP2C18-1F (described above)

CYP2C19-1F (described above)

CYP2C19-1R (described above)

hloxP-SacII-EcoRI-F (described above)

PGKr1 (described above)

hloxP-1R (described above)

TRANS L1 (described above)

TRANS R1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,nine clones out of the 11 clones were found to be positive for allprimer sets, and the following analysis was carried out by using thosenine clones.

[D. 2. 2] Two-Color FISH Analysis

With the nine clones of CHO (CYP2C-MAC, hChr10-ΔCYP2C) obtained from theabove, FISH analysis was carried out by using CYP2C-BAC(RP11-466J14)(CHORI) DNA and mouse Cot-1 DNA as probes according to the methoddescribed by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175,2001). As a result, it was confirmed that the signal derived from humanCYP2C was observed on MAC4 at a rate of 50% or more in three clones outof the nine clones (FIG. 94).

From these results, it was concluded that 380 kb of CYP2C cluster onhuman chromosome 10 fragment can be cloned into the mouse artificialchromosome vector MAC4 by reciprocal translocation.

[E] Transfer of CYP2C-MAC from CHO Cell to Mouse A9 Cell

To prepare mouse ES cells retaining CYP2C-MAC, introduction was carriedout from CHO cells (CHO (CYP2C-MAC, hChr10-ΔCYP2C) 2, 8, 10) retainingCYP2C-MAC obtained from the above [D] to, as a mouse A9 cell, mouse A9cells having high microcell forming ability by microcell fusion. Totalfour resistant colonies obtained by four microcell fusions wereisolated, amplified, and subjected to the following analysis (clonename: A9 (CYP2C-MAC)). As a result, there were four clones which arepositive in PCR using the primers described before for detecting theCYP2C-MAC region only. In addition, FISH analysis (Tomizuka et al.,Nature Genet. 16: 133, 1997) was carried out by usingCYP2C-BAC(RP11-466J14) (CHORI) and mouse minor satellite DNA as probes.As a result, the presence of CYP2C-MAC, which is specifically detectedwith the probes, was confirmed in two clones out of the four clones.From the above, it was concluded that two clones of A9 cells retainingCYP2C-MAC are obtained.

[F] As described in Example 8, in vitro stability can be examined bypreparing mouse ES cells retaining the mouse artificial chromosomevector CYP2C-MAC and using it. Further, by preparing a chimeric mouseusing the ES cells, the mouse lineage-based TC(CYP2C-MAC) in whichCYP2C-MAC is transferred to a progeny can be prepared. Further, by usingthe TC(CYP2C-MAC) mouse line, stability of CYP2C-MAC in somatic cellscan be examined. Still further, the liver microsome derived fromTC(CYP2C-MAC) mouse line may be used as a sample for testing apharmacological effect and toxicity in the phase I reaction fordevelopment of a pharmaceutical product. Further, because human drugmetabolism can be reproduced by TC(CYP2C-MAC) mouse line, it may be alsoused as a model mouse for in vivo test that is used for testing apharmacological effect and toxicity in the phase I reaction fordevelopment of a pharmaceutical product.

Example 20 Construction of the Mouse Artificial Chromosome VectorMDR1-MAC

Translocation cloning of MDR1 gene, which is a human drug metabolizingenzyme gene group, into the mouse artificial chromosome vector MAC4 isperformed by using Cre/loxP system to construct MDR1-MAC in the samemanner as in Example 3.

[A] Site Specific Insertion of loxP Sequence into AC005045 on HumanChromosome 7

For translocation insertion to the mouse artificial chromosome vectorMAC4 via loxP sequence, loxP sequence is inserted into AC005045 proximalto MDR1 gene of human chromosome 7 (hChr7) in DT40 cells.

[A. 1] Preparation of Targeting Vector pMDR1loxPbs

Targeting vector pMDR1loxPbs for inserting loxP as a recognitionsequence for Cre recombinase into AC005045 region, which is locatedextremely close to MDR1 gene locus of human chromosome 7 and on thecentromere side (i.e., locating on the centromere side by approximately50 Kb from MDR1 gene locus), was prepared as follows. First, theAC005045 genome region was amplified by PCR using the following primers.

MDR1loxP2L: (SEQ ID NO: 184) 5′- gccaagtgtagctggagaatgattcgtg -3′MDR1loxP1R: (SEQ ID NO: 185) 5′- acaaggcacttcaggataccaagcttcc -3′

As a basic plasmid for inserting loxP sequence, V901 (Lexicon genetics)was used. For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. wasused as a thermal cycler and LA Taq (TAKARA SHUZO CO., LTD.) was used asTaq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used werethose included in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 7 min were carried out.PCR product was treated with proteinase K (Gibco) and subjected to gelfiltration by using CHROMASPIN-TE400 (Clontech). After that, the productwas cleaved with the restriction enzyme BglII (NIPPON GENE CO., LTD.)and subjected to gel filtration by using CHROMASPIN-TE1000 (Clontech).The PCR fragments (2.5 kb and 5.3 kb) were cloned into the BglII andBamHI sites of V901 plasmid (vector name: V901-MDR1HR2). Next,V901-MDR1HR2 was cleaved with the restriction enzymes AscI (NEB) andKpnI and the DNA fragment containing loxP was cut out from the cassettevector Bs-loxP-3′ HPRT (Hoshiya et al., Mol. Ther. 2009; 17(2): 309-17)by using the restriction enzymes AscI and KpnI, and then they wereligated to each other. The resultant product having the loxP sequence inthe same direction as the cloned AC005045 genome fragment was taken astargeting vector pMDR1loxPbs. Size of the final construct inserted withloxP is 13.0 kb. The targeting vector, target sequence, and chromosomeallele obtained by homologous recombination are shown in FIG. 95.

[A. 2] Transfection and Isolation of Drug Resistant Clone

As described above, the targeting vector pMDR1loxPbs prepared above waslinearized with the restriction enzyme NotI (TAKARA), and used fortransfection of chicken DT40 cells (cloneDT40-#7) retaining humanchromosome 7, which is prepared according to the method described in WO01/011951. After exchanging the culture medium for culture mediumcontaining blasticidin S (15 μg/ml), the cells were dispensed into three96-well culture plates and then subjected to selection culture for about2 weeks. Total nine resistant colonies obtained by two transfectionswere isolated, amplified, and subjected to the following analysis (clonename: DT40 (hChr7M-loxP)).

[A. 3] Selection of Homologous Recombinant

[A. 3. 1] PCR Analysis

Genomic DNA was extracted from the blasticidin S resistant clones byusing Puregene DNA Isolation Kit (Gentra Systems, Inc.) andidentification of homologous recombinant was carried out by PCR usingthe following two sets of primer.

Identification of homologous recombinant was carried out by PCR usingthe following two sets of primers.

MDR1loxP2L (described above) (SEQ ID NO: 186) BsdR:5′-gctcaagatgcccctgttct-3′ (SEQ ID NO: 187) hprt332F:5′-aaagatggtcaaggtcgcaa-3′ MDR1loxP1R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 10 sec and 68° C. for 4 min were carried out. As a result ofscreening nine clones, three clones were identified as a homologousrecombinant.

[A. 3. 3] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). With the three clones which havebeen confirmed to have recombination in the above, FISH analysis wascarried out by using human cot-1 DNA and blasticidin DNA as probes. As aresult, it was found that human chromosome 7 was not translocated to thehost chromosome in any clone, and based on the fact thatneomycin-derived signal was detected near 7q21, it was confirmed thatrecombination has site-specifically occurred. From these results, it wasconcluded that loxP sequence as a gene introduction site wassite-specifically inserted into AC005045 of human chromosome 7.

[B] Site Specific Cleavage at AC003083 on Human Chromosome 7

To delete the gene at the distal side from MDR1 gene of human chromosome7, telomere truncation as site specific deletion of chromosome isperformed.

[B. 1] Preparation of Targeting Vector pTELpuro-MDR1

Targeting vector pTELpuro-MDR1 for inserting human telomere sequenceinto AC003083 region, which was located extremely close to MDR1 genelocus of human chromosome 7 and on the telomere side (i.e., locating onthe telomere side by approximately 50 Kb from MDR1 gene locus), wasprepared as follows. First, the AC003083 genome region was amplified byPCR using the following primers.

MDR1tel5L; (SEQ ID NO: 188) 5′- ctattctaaaaagctgccttggcccaca-3′MDR1tel5R; (SEQ ID NO: 189) 5′- tgtagcccagttcctaatgggacacaga-3′

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 35 cycles of 98°C. for 20 sec and 68° C. for 8 min were carried out. PCR product wastreated with proteinase K (Gibco) and subjected to gel filtration byusing CHROMASPIN-TE400 (Clontech). After that, the product was cleavedwith the restriction enzymes EcoRI (NIPPON GENE CO., LTD.) and PstI(NIPPON GENE CO., LTD.) and subjected to gel filtration by usingCHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the EcoRIand PstI sites of plasmid pTELpuro (Kuroiwa et al., Nature Biotech., 20:88, 2002). Since the genome sequence of AC003083 is in the direction ofcentromere→telomere, the resultant in which cloned AC003083 genomefragment is in the same direction as the human telomere sequence wastaken as desired targeting vector pTELpuro-MDR1. Size of the finalconstruct for long-arm proximal region specific restriction was 13.1 kb.The targeting vector, target sequence, and chromosome allele obtained byhomologous recombination are shown (FIG. 96).

[B. 2] Transfection and Isolation of Drug Resistant Clone

As described above, the targeting vector pTELpuro-MDR1 prepared abovewas linearized with the restriction enzyme EcoRI (NIPPON GENE CO.,LTD.), and used for transfection of the clone DT40 (hChr7M-loxP) 8, 9prepared above. After exchanging the culture medium for culture mediumcontaining puromycin (0.3 μg/ml), the cells were dispensed into ten96-well culture plates and then subjected to selection culture for about2 weeks. Total 96 resistant colonies obtained by four transfections wereisolated, amplified, and subjected to the following analysis (clonename: DT40 (hChr7M-loxP-tel)).

[B. 3] Selection of Homologous Recombinant

[B. 3. 1] PCR Analysis

In order to select a recombinant by using as a template the genomic DNAof puromycin resistant cell line, as a primary screening, PCR wascarried out by using the following primers that are located closer tothe telomere side than the restriction sites, and occurrence of sitespecific cleavage was examined. The primer sequences are given below.

CYP3A4 R (described above)

CYP3A4 F (described above)

CYP3A5 R (described above)

CYP3A5 F (described above)

CYP3A7 R (described above)

CYP3A7 F (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and Ampli Taq Gold (Applied Biosystems) was used as Taqpolymerase. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 95° C. for 10min, 30 cycles of 95° C. for 20 sec, 55° C. for 30 sec, and 72° C. for30 sec were carried out.

Next, for three clones that are not detected with the above primers, itwas confirmed, by PCR using the following primers, whether or not sitespecific homologous recombination has occurred. Sequences are asfollows.

MDR1tel5L; (SEQ ID NO: 190) 5′-ATCTGCAGGGAAGGGATCCAGTTTCAGCTTCCTAC-3′SK23 (described above) MDR1-1L: (SEQ ID NO: 191)5′-ctcctaggagtactcacttc-3′ MDR1-1R: (SEQ ID NO: 192)5′-aacagaaacatggcttggcg-3′ MDR1-2L: (SEQ ID NO: 193)5′-cgccaagccatgtttctgttt-3′ MDR1-2R: (SEQ ID NO: 194)5′-aaggaaatgctttctgccttg-3′ MDR1-3L: (SEQ ID NO: 195)5′-gtgcaacggaagccagaaca-3′ MDR1-3R: (SEQ ID NO: 196)5′-agcggcctctgcttctttga-3′ MDR1-4L: (SEQ ID NO: 197)5′-ctgattggctgggcaggaac-3′ MDR1-4R: (SEQ ID NO: 198)5′-cttggaacggccaccaagac-3′ MDR1-5L: (SEQ ID NO: 199)5′-ggtgctggttgctgcttaca-3′ MDR1-5R: (SEQ ID NO: 200)5′-cccaacatcgtgcacatcaa-3′ MDR1-6L: (SEQ ID NO: 201)5′-gtcagtgttgatggacagga-3′ MDR1-6R: (SEQ ID NO: 202)5′-gcattggcttccttgacagc-3′ MDR1-7L: (SEQ ID NO: 203)5′-ggttccaggcttgctgtaat-3′ MDR1-7R: (SEQ ID NO: 204)5′-tctttcagtgcttgtccaga-3′ MDR1-8L: (SEQ ID NO: 205)5′-ggcaaagaaataaagcgactg-3′ MDR1-8R: (SEQ ID NO: 206)5′-cctcctttgctgccctcaca-3′ MDR1-9L: (SEQ ID NO: 207)5′-tcttgtccaaactgcctgtga-3′ MDR1-9R: (SEQ ID NO: 208)5′-tgcaagaatcagcaggatcaa-3′

For PCR, LA Taq (TAKARA SHUZO CO., LTD.) was used with the aboveprimers. Buffers and dNTPs (dATP, dCTP, dGTP, dTTP) used were thoseincluded in the product and they were used under the conditionsdescribed by manufacturer's instruction. Temperature and cycleconditions were as follows: after heat denaturation at 94° C. for 1 min,35 cycles of 98° C. for 20 sec and 68° C. for 8 min were carried out.Only in three clones having site specific recombination, a band atapproximately 8 kb was detected. In DT40 and DT40 (hChr7M-loxP) 8, 9 asnegative controls, no band was detected.

[B. 3. 2] Two-Color FISH Analysis

FISH analysis was carried out according to Matsubara et al. (FISH testprotocol, Shujunsha Co., Ltd., 1994). With the three clones which havebeen confirmed to have recombination in the above, FISH analysis wascarried out by using human cot-1 DNA and puromycin DNA as probes. As aresult, it was found that human chromosome 7 was not translocated to thehost chromosome in any clone, and based on the fact thatpuromycin-derived signal was detected at terminal of human chromosome 7fragment and restrictions occurred at desired sites, it was confirmedthat recombination has site-specifically occurred (FIG. 97).

From these results, it was concluded that, in clone DT40(hChr7M-loxP-tel) 10, 12, and 70, cleavage could be made at distalregion from AC003083 which was closer to the telomere side than MDR1gene region.

[C] Transfer of hChr7M-loxP-tel from DT40 Containing hChr7M-loxP-tel toCHO Cell Containing MAC4

For translocation insertion of human MDR1 gene region into the mouseartificial chromosome vector MAC4 via loxP sequence in CHO cells,hChr7M-loxP-tel is introduced into CHO cells containing the mouseartificial chromosome vector MAC4.

[C. 1] Microcell Fusion and Isolation of Drug Resistant Clone

By using DT40 (hChr7M-loxP-tel) 10 and 70 as recipient cells, microcellfusion was carried out for CHO(HPRT⁻; MAC4), which is a CHO hprtdepleted cell containing MAC4 (obtained from the Health Science ResearchResources Bank, registration number: JCRB0218), in the same manner asabove. Total 15 resistant colonies obtained by four microcell fusionswere isolated, amplified, and subjected to the following analysis (clonename: CHO(HPRT⁻; MAC4, hChr7M-loxP-tel)).

[C. 2] Selection of Drug Resistant Clone

[C. 2. 1] PCR Analysis

For extracting genomic DNA from blasticidin S resistant cell line andusing it as a template for selecting a recombinant, PCR was carried outby using the following primers and it was confirmed whether or not humanchromosome 7 fragment was introduced into CHO cells containing MAC4. Theprimer sequences are given below.

m11 4L: (described above) V907-NotI-R: (described above) hygF (244):(described above) ml1 6R (described above) MDR1loxP2L (described above)(SEQ ID NO: 209) BsdR: 5′-gctcaagatgcccctgttct-3′ (SEQ ID NO: 210)hprt332F: 5′-aaagatggtcaaggtcgcaa-3′ MDR1loxP1R (described above)MDR1tel5L (described above) SK23 (described above) MDR1-1L(described above) MDR1-1R (described above) MDR1-2L (described above)MDR1-2R (described above) MDR1-3L (described above) MDR1-3R(described above) MDR1-4L (described above) MDR1-4R (described above)MDR1-5L (described above) MDR1-5R (described above) MDR1-6L(described above) MDR1-6R (described above) MDR1-7L (described above)MDR1-7R (described above) MDR1-8L (described above) MDR1-8R(described above) MDR1-9L (described above) MDR1-9R (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,six clones out of the 15 clones were found to be positive for all primersets, and the following analysis was performed by using those 6 clones.

[C. 2. 2] Two-Color FISH Analysis

With the six clones of CHO(HPRT⁻; MAC4, hChr7M-loxP-tel) obtained fromthe above, FISH analysis was carried out by using mouse Cot-1 DNA andhuman Cot-1 DNA as probes according to the method described by Shinoharaet al. (Human Molecular Genetics, 10: 1163-1175, 2001). As a result, itwas confirmed that one or two copies of MAC1 and hChr7M-loxP-tel wereintroduced into CHO cells at a rate of 80% or more in two clones (FIG.98).

From these results, it was concluded that hChr7M-loxP-tel could beintroduced into CHO cells containing the mouse artificial chromosomevector MAC4.

[D] Site Specific Translocation of 210 kb Human MDR1 Gene Region (i.e.,AC005045-Human MDR1 Gene-AC003083) to MAC4 Vector in CHO(HPRT⁻; MAC4,hChr7M-loxP-tel) Clone

To stably keep the human MDR1 gene, which is a 210 kb DNA, in a mouseindividual, translocation insertion into the mouse artificial chromosomevector MAC4 is performed (FIG. 99).

[D. 1] Transfection and Isolation of HAT Resistant Clone

Gene introduction was carried out by lipofection for CHO(HPRT⁻; MAC4,hChr7M-loxP-tel) 7 and 15 obtained from the above. To cells in 6 wellswith 90% confluency, 3 μg of Cre was introduced according to thecommercially available protocol (Invitrogen). After culture for 2 weeksunder HAT selection culture, a resistant colony was generated and totalten colonies obtained by two introductions were isolated, amplified, andsubjected to the following analysis (clone name: CHO (MDR1-MAC,hChr7-ΔMDR1)).

[D. 2] Selection of Drug Resistant Clone

[D. 2. 1] PCR Analysis

For extracting genomic DNA from HAT resistant cell line and using it asa template for selecting a clone with reciprocal translocation, PCR wascarried out by using the following primers and it was confirmed whetheror not reciprocal chromosomal translocation has occurred on humanchromosome 7 fragment and MAC4. The primer sequences are given below.

m11 4L: (described above) V907-NotI-R: (described above) hygF (244):(described above) ml1 6R (described above) MDR1loxP2L (described above)(SEQ ID NO: 211) BsdR: 5′-gctcaagatgcccctgttct-3′ (SEQ ID NO: 212)hprt332F: 5′-aaagatggtcaaggtcgcaa-3′ MDR1loxP1R (described above)MDR1tel5L (described above) SK23 (described above) MDR1-1L(described above) MDR1-1R (described above) MDR1-2L (described above)MDR1-2R (described above) MDR1-3L (described above) MDR1-3R(described above) MDR1-4L (described above) MDR1-4R (described above)MDR1-5L (described above) MDR1-5R (described above) MDR1-6L(described above) MDR1-6R (described above) MDR1-7L (described above)MDR1-7R (described above) MDR1-8L (described above) MDR1-8R(described above) MDR1-9L (described above) MDR1-9R (described above)TRANS L1 (described above) TRANS R1 (described above)

For PCR, GeneAmp 9600 manufactured by PerkinElmer, Inc. was used as athermal cycler and LA Taq (TAKARA) was used as Taq polymerase. Buffersand dNTPs (dATP, dCTP, dGTP, dTTP) used were those included in theproduct and they were used under the conditions described bymanufacturer's instruction. Temperature and cycle conditions were asfollows: after heat denaturation at 94° C. for 1 min, 30 cycles of 98°C. for 10 sec and 68° C. for 7 min were carried out. As a result of PCR,six clones out of ten clones were found to be positive for all primersets, and the following analysis was performed by using those sixclones.

[D. 2. 2] Two-Color FISH Analysis

With the six clones of CHO (MDR1-MAC, hChr7-ΔMDR1) obtained from theabove, FISH analysis was carried out by using MDR1-BAC(RP11-784L5)(CHORI) DNA and mouse Cot-1 DNA as probes according to the methoddescribed by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175,2001). As a result, it was confirmed that the signal derived from humanMDR1 was observed on MAC4 at a rate of 60% or more in three clones outof the six clones (FIG. 100).

From these results, it was concluded that 210 kb of MDR1 gene of humanchromosome 7 fragment could be cloned into the mouse artificialchromosome vector MAC4 by reciprocal translocation.

[E] Transfer of MDR1-MAC from CHO Cells to Mouse A9 Cells

To prepare mouse ES cells retaining MDR1-MAC, transfer was carried outfrom CHO cells retaining MDR1-MAC obtained from the above [D] (CHO(MDR1-MAC, hChr7-ΔMDR1) 1, 2, 4) to, as mouse A9 cells, mouse A9 cellshaving high microcell forming ability by microcell fusion. Total sevenresistant colonies obtained by four microcell fusions were isolated,amplified, and subjected to the following analysis (clone name: A9(MDR1-MAC)). As a result, there were five clones which are positive inPCR using the primers described before for detecting the MDR1-MAC regiononly. In addition, FISH analysis (Tomizuka et al., Nature Genet. 16:133, 1997) was carried out by using MDR1-BAC(RP11-784L5) (CHORI) andmouse minor satellite DNA as probes. As a result, the presence ofMDR1-MAC region, which is specifically detected with the probes, wasconfirmed in three clones out of the five clones. From these results, itwas concluded that three clones of A9 cells retaining MDR1-MAC wereobtained.

[F] As described in Example 8, in vitro stability can be examined bypreparing mouse ES cells retaining the mouse artificial chromosomevector MDR1-MAC. Further, by preparing a chimeric mouse using the EScells, the mouse line-based TC (MDR1-MAC) in which MDR1-MAC istransferred to a progeny can be prepared. Further, by using the TC(MDR1-MAC) mouse line, stability of MDR1-MAC in somatic cells can beexamined. Further, TC (MDR1-MAC) mouse line allows reproduction of drugtransport in human or the like. Still further, sinceTC(CYP3A-MAC/MDR1-MAC) line can be prepared by crossbreeding with theTC(CYP3A-MAC) mouse line, it may be used as a model mouse for in vivotest that is used for testing a pharmacological effect and toxicity fordevelopment of a pharmaceutical product.

INDUSTRIAL APPLICABILITY

The mouse artificial chromosome vector of the invention has the sameusefulness as the human artificial chromosome described in WO2009/063722. Further, having enhanced retention rate in rodent cells orindividuals, it can be stably retained in rodent cells to retain stablythe target gene (group) for a long period of time. Still further, asthere is no deviation in amount of introduced gene among individuals ortissues of rodents like a mouse, more accurate analysis of introducedgene can be achieved among individuals or tissues. The mouse artificialchromosome vector of the invention can be used for various purposes anduses like introduction of an exogenous gene to a recipient cell,establishment of iPS cells or use for regenerative medicine, preparationof cells for expressing exogenous genes or useful non-human animals,protein production, and analysis of gene function.

Accession number of Deposit:

Accession number of DT40 B6bT-1: FERM BP-11128

Free text of sequence listings

SEQ ID NO: 1 to 212: Primer

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

The invention claimed is:
 1. A mouse artificial chromosome vector,comprising: a natural centromere derived from a mouse chromosome 11; amouse-chromosome-derived long-arm fragment formed by deleting at leastabout 99% of all endogenous genes from the long-arm distal regionproximal to the centromere of mouse chromosome 11; a telomere sequenceso as to form the mouse artificial chromosome vector contained in thedeposited cell line DT40 B6bT-1 (FERM BP-11128) wherein the vector isstably retained in cells and tissues of a mammal at a retention rate ofabout 90% or more.
 2. The mouse artificial chromosome vector accordingto claim 1, wherein the mouse-chromosome-derived long-arm fragment isthe remainder formed by deleting a region including at least 99.5% ofall endogenous genes from a long arm of mouse chromosome
 11. 3. Themouse artificial chromosome vector according to claim 1, wherein themammal is a rodent.
 4. The mouse artificial chromosome vector accordingto claim 3, wherein the rodent is a mouse, rat, or hamster.
 5. The mouseartificial chromosome vector according to claim 1, further comprisingone or more DNA sequence insertion sites.
 6. The mouse artificialchromosome vector according to claim 5, wherein the DNA sequenceinsertion site is a recognition site for a site-specific recombinase. 7.The mouse artificial chromosome vector according to claim 5, wherein theDNA sequence insertion site is a loxP sequence, an FRT sequence, φC31attB and φC31 attP sequences, R4 attB and R4 attP sequences, TP901-1attB and TP901-1 attP sequences, or Bxb1 attB and Bxb1attP sequences. 8.The mouse artificial chromosome vector according to claim 1, furthercomprising a reporter gene, a selection marker gene, or both.
 9. Themouse artificial chromosome vector according to claim 1, furthercomprising an exogenous DNA sequence.
 10. The mouse artificialchromosome vector according to claim 1, wherein the exogenous DNAsequence has a size of 200 kb or more.
 11. The mouse artificialchromosome vector according to claim 9, wherein the exogenous DNAsequence is a human DNA sequence.
 12. The mouse artificial chromosomevector according to claim 9, wherein the exogenous DNA sequence is a DNAsequence of a drug-metabolism-related gene.
 13. The mouse artificialchromosome vector according to claim 12, wherein thedrug-metabolism-related gene is a gene encoding an enzyme involved in aphase I reaction or a phase II reaction.
 14. The mouse artificialchromosome vector according to claim 13, wherein the enzyme geneinvolved in a phase I reaction encodes at least one enzyme selected fromthe group consisting of CYPs of CYP1A, CYP1B, CYP2A, CYP2B, CYP2C,CYP2D, CYP2E, CYP2J, CYP3A, CYP4A, CYP4B and subfamilies thereof, andCESs.
 15. The mouse artificial chromosome vector according to claim 13,wherein the enzyme gene involved in the phase II reaction encodes atleast one enzyme selected from the group consisting of UGT1 and UGT2.16. The mouse artificial chromosome vector according to claim 12,wherein the drug-metabolism-related gene is a gene encoding atransporter.
 17. The mouse artificial chromosome vector according toclaim 16, wherein the gene encoding the transporter is at least one geneselected from the group consisting of MDR1, MDR2, MRP2, OAT, OATP, OCT,and BCRP.
 18. The mouse artificial chromosome vector according to claim12, wherein the drug-metabolism-related gene is a gene encoding anuclear receptor.
 19. The mouse artificial chromosome vector accordingto claim 18, wherein the gene encoding the nuclear receptor is at leastone gene selected from the group consisting of PXR, AhR, CAR, and PPARα.20. The mouse artificial chromosome vector according to claim 9, whereinthe exogenous DNA sequence is a DNA sequence of ahuman-chromosome-derived long arm or short arm.
 21. The mouse artificialchromosome vector according to claim 9, wherein the exogenous DNAsequence comprises at least two genes selected from the group consistingof genes encoding an enzyme involved in a phase I reaction, genesencoding an enzyme involved in a phase II reaction, genes encoding atransporter, and genes encoding a nuclear receptor.
 22. The mouseartificial chromosome vector according to claim 20, wherein the DNAsequence of the human-chromosome-derived long arm or short arm comprisesa human chromosome region responsible for a disease gene.
 23. The mouseartificial chromosome vector according to claim 9, wherein the exogenousDNA sequence is a gene or DNA sequence encoding a polypeptide selectedfrom the group consisting of cytokines, hormones, growth factors,nutritional factors, hematopoietic factors, coagulation or hemolysisfactors, immunoglobulins, G protein-coupled receptors, or enzymes, or agene or DNA sequence used for treatment involved in a disease electedfrom the group consisting of muscular dystrophy, hemophilia,neurodegenerative disease, autoimmune disease, allergic disease, orgenetic disease.
 24. The mouse artificial chromosome vector according toclaim 1, wherein the cell is a hepatocyte, enterocyte, renal cell,splenocyte, lung cell, cardiac cell, skeletal muscle cell, brain cell,bone marrow cell, lymphocyte, megakaryocyte, sperm, or ovum.
 25. Themouse artificial chromosome vector according to claim 1, wherein thetissue is from a liver, intestine, kidney, spleen, lung, heart, skeletalmuscle, brain, bone marrow, testis, or ovary.
 26. An isolated cellcomprising the mouse artificial chromosome vector according to claim 1.27. The cell according to claim 26, wherein the cell is selected fromthe group consisting of somatic cells, non-human germ-line cells, stemcells, and precursor cells.
 28. The cell according to claim 27, whereinthe stem cell is an embryonic stem (ES) cell or an induced pluripotentstem (iPS) cell.
 29. The cell according to claim 26, wherein the cell isa primary cultured cell, subcultured cell, or cell line.
 30. The cellaccording to claim 26, wherein the cell is capable of producing a humanantibody.
 31. A pharmaceutical composition comprising the isolated cellaccording to claim 26, wherein the cell comprises a mouse artificialchromosome vector comprising an exogenous DNA sequence for use intreating a disease.
 32. A process for producing a protein, comprising:culturing the isolated cell according to claim 26, comprising the mouseartificial chromosome vector comprising a sequence of an exogenous DNAto produce a protein encoded by the DNA; and collecting the protein.